Method and apparatus for bulk extraction and high-level purification of extracellular vesicles from biological samples

Abstract

The present invention relates to a method and apparatus for bulk extraction and high-level purification of extracellular vesicles from biological samples. More specifically, in order to separate and extract the extracellular vesicles from large amount of culture medium, extracellular vesicles are adsorbed and extracted as same pass through a filter made of a porous medium surface-modified with a cationic substance. According to the present invention, exosomes or extracellular vesicles can be economically and efficiently separated and purified to a high level, and extracellular vesicles can be separated and purified in bulk. Therefore, the present invention is suitable for GMP and makes it possible to economically and efficiently separate and purify exosomes or extracellular vesicles of high purity and uniform particle size distribution at high yield.

Description

Method and device for extracting extracellular vesicles in large quantities from biological samples and purifying them with high purity

[0001] The present invention relates to a method and apparatus for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, and more particularly, to a method and apparatus for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, which comprises passing the extracellular vesicles through a filter composed of a porous medium whose surface is modified with a cationic substance and a tangential flow filtration device connected in series to separate and extract the extracellular vesicles from a large volume of culture medium.

[0002] Recently, studies have reported that cell secretions (secretome) contain various bioactive factors that control cell behavior, and in particular, cell secretions contain 'exosomes' or 'extracellular vesicles (EVs),' which are nano-vesicles with intercellular signaling functions, and research on their components and functions is actively underway.

[0003] Cells release various membrane-bound vesicles into the extracellular environment, commonly referred to as extracellular vesicles. Extracellular vesicles are also known by other names, including membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, and exosomes, and are sometimes used interchangeably with exosomes.

[0004] In the present invention, microvesicles and exosomes are collectively defined as extracellular vesicles.

[0005] Exosomes are vesicles measuring tens to hundreds of nanometers in size, composed of a double phospholipid membrane with the same structure as the cell membrane. They contain proteins, mRNA, miRNA, and other components known as exosome cargo. Exosome cargo contains a wide range of signaling factors, which are known to be cell-type specific and differentially regulated depending on the secretory cell's environment.

[0006] Exosomes are intercellular signaling mediators secreted by cells, and various cell signals transmitted through them are known to regulate cell behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.

[0007] Exosomes contain specific genetic material and bioactive factors, depending on the nature and state of the cells from which they originate. Exosomes derived from proliferating stem cells regulate cell behaviors such as migration, proliferation, and differentiation, and reflect the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

[0008] Conventional techniques for isolating exosomes or extracellular vesicles include ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, polymer-based precipitation, and tangential flow filtration.

[0009] Ultracentrifugation has been the most widely used method to isolate exosomes or extracellular vesicles. However, it has drawbacks such as low yield, time-consuming separation, labor-intensive nature, and the need for expensive equipment. Furthermore, ultracentrifugation can damage exosomes or extracellular vesicles during the separation process, potentially interfering with subsequent analysis or applications.

[0010] Ultrafiltration can be used in conjunction with ultracentrifugation to increase the purity of exosomes or extracellular vesicles, but has the problem that the exosomes or extracellular vesicles stick to the filter, resulting in a low yield after separation.

[0011] Immunoaffinity separation method has the advantage of high specificity as it is a method of isolating antibodies by attaching them to exosomes or extracellular vesicles, but it has the disadvantage of requiring a process for producing antibodies and a process for removing antibodies after separation, which is expensive and unsuitable for scale-up.

[0012] Meanwhile, various exosome isolation kits, such as exosome precipitation, total exosome isolation kit, or polymer-based precipitation, have been commercially sold recently as a method for isolating exosomes. However, although they are easy to use, the reagents are expensive, so although they can be used to isolate exosomes or extracellular vesicles at the laboratory level, they are not suitable for isolating and purifying exosomes or extracellular vesicles in large quantities.

[0013] Tangential Flow Filtration (TFF) is a method of filtering target substances by moving fluid parallel to the membrane filter surface. It has the advantage of being able to process large volumes of samples in a continuous process, but the recovery rate is known to be very low compared to the previously used technology, sedimentation.

[0014] Above all, the problem in the process of isolating exosomes or extracellular vesicles is that impurities such as proteins are mixed in and separated together, resulting in low purity and yield.

[0015] Additionally, the isolation and purification of exosomes or extracellular vesicles is time-consuming, cumbersome, and expensive.

[0016] Additionally, existing separation methods developed to increase purity have problems with scale-up being difficult and not suitable for Good Manufacturing Practice (GMP).

[0017] Accordingly, in the technical field to which the present invention pertains, there is a persistent demand for a technology that can economically and efficiently isolate and purify exosomes or extracellular vesicles with high purity, while also enabling scale-up and being suitable for Good Manufacturing Practice (GMP). In other words, there is a need for a new technology that can economically and efficiently isolate and purify exosomes or extracellular vesicles with high purity and uniform particle size distribution, with high yield.

[0018] The present invention, which aims to solve the above-mentioned problems, provides a method and device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity by fusing a technology for separating them by attaching cationic substances to a capture surface such as a microparticle or a membrane by utilizing the negative charge characteristics and size of 30 nm to 500 nm of extracellular vesicles and exosomes produced in a large-scale extracellular vesicle culture solution, and a technology for releasing only substances of sub-nanometer size.

[0019] In addition, the present invention aims to provide a method and device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, which dramatically increases the capture efficiency of extracellular vesicles by constructing a filter with a porous medium of a microstructure to increase the surface area to which extracellular vesicles are attached.

[0020] In addition, the present invention aims to provide a method and device for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, by processing a large volume of culture solution in a continuous process by configuring a device composed of a porous medium in the form of a filter, thereby improving the efficiency of extraction and purification of extracellular vesicles.

[0021] In addition, the purpose is to provide a method and device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity by filtering extracellular vesicles using a filter arranged vertically with respect to the direction of movement of the culture medium and removing residual substances such as proteins having a small particle size or molecular weight using a tangential flow filtration device arranged horizontally with respect to the direction of movement of the culture medium, thereby extracting highly pure extracellular vesicles.

[0022] The present invention, which has been devised to solve the above-mentioned problems, is a method for extracting extracellular vesicles from an extracellular vesicle solution, comprising: a first step of preliminarily filtering the extracellular vesicle solution; a second step of introducing the filtered extracellular vesicle solution into a microfilter structure composed of a microporous structure surface-modified with a positively charged substance to adsorb the extracellular vesicles; a third step of introducing a sample solution passed after the second step into a tangential flow filtration device to remove foreign substances below a certain size; a fourth step of repeating the second and third steps a predetermined number of times; and a fifth step of injecting an eluent into the microfilter structure to separate the extracellular vesicles.

[0023] The above microfilter structure is characterized in that it is formed of a microporous structure of any one of microparticles, a membrane structure, a mesh structure, and a laminated structure of spherical beads.

[0024] The above microfilter structure is characterized in that it is installed inside a syringe storing the extracellular vesicle solution.

[0025] The surface of the above microporous structure is characterized by being modified by a polycationic substance.

[0026] The above polycationic material is characterized by being at least one selected from the group consisting of polylysine, protamine, polyarginine, polyhistidine, cationic dextran, cationic dendrimer, cationic polysaccharide, polyamidoamine, polyethyleneimine, polyquaternium, poly-2-dimethylaminoethyl methacrylate (PDMAEMA), poly-2-dimethylaminomethyl styrene (PDMAMS), poly-1-vinylpyrrolidone (p1-VP), poly diethylaminoethyl acrylate (pDEAEA), poly dimethylaminoethyl acrylate (pDMAEA), poly diethylaminoethyl methacrylate (pDEAMA), lipopolyamines, quaternary ammoniums, guanidine, imidazole, polyaniline, polypyrrol, and chitosan.

[0027] The microparticles included in the above microporous structure are characterized by being composed of at least one of polyacrylate, polyacrylamide, polymethacrylate, polyethylene glycol, polystyrene vinyl benzene, polystyrene, hydrogel, agarose, ceramic, silica, latex, metal particles, glass particles, and magnetic particles.

[0028] The fifth step of separating the extracellular vesicles by injecting the eluent includes the fifth-first step of injecting the first reagent into the microfilter structure and washing the microfilter structure under the first condition, and the first reagent is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH - A material including at least one of the following, wherein the first condition is set to be performed at least once within a pH range of at least one of pH 5.5 to 6.5 and pH 7.5 to 9.5.

[0029] After the above step 5-1, a second reagent is added to the microfilter structure, and a step 5-2 is further included to wash the microfilter structure under a second condition, and the second reagent is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH - A substance having a concentration of 0.2 M to 3 M, which includes at least one of the above, and the second condition is optionally set to be performed at least once while mixing at 500 rpm to 2000 rpm for a time of 1 minute to 60 minutes.

[0030] It is characterized in that a step 5-3 for performing additional buffer substitution is further included after the step 5-2, and the buffer substituted in the step 5-3 is at least one of physiological saline solution, deionized water, and distilled water.

[0031] According to another embodiment, the present invention provides a device for extracting extracellular vesicles from an extracellular vesicle solution, comprising: a first syringe; a second syringe; a tangential flow filtration device having both ends connected to outlets of the first syringe and the second syringe, respectively; an outlet formed at one side of the tangential flow filtration device; an outlet container connected to the outlet; and a microfilter structure installed inside at least one of the first syringe and the second syringe.

[0032] The extracellular vesicle solution injected into the first syringe is characterized in that it passes through the tangential flow filtration device and then flows into the second syringe.

[0033] The above extracellular vesicle solution is characterized in that it moves by positive pressure inside the first syringe or negative pressure inside the second syringe.

[0034] The above microfilter structure is characterized in that it filters the extracellular vesicles contained in the extracellular vesicle solution by adsorbing them on the surface of a microporous structure surface-modified with a positively charged substance.

[0035] The above tangential flow filtration device has a structure in which a hollow fiber membrane is formed in a direction that is horizontal to the direction in which the extracellular vesicle solution flows or is spread at an angle less than 90°, and is characterized in that foreign substances are discharged through microscopic pores formed in the hollow fiber membrane.

[0036] The size of the pores formed in the hollow fiber membrane is characterized by being 50 nm or less.

[0037] The size of the pores formed inside the above microporous structure is characterized by being 800 nm or more.

[0038] According to another embodiment, the present invention provides a device for extracting extracellular vesicles from an extracellular vesicle solution, comprising: a supply container for storing an extracellular vesicle solution including extracellular vesicles to be separated, and supplying the stored solution; a microfilter structure for filtering extracellular vesicles contained in the extracellular vesicle solution by adsorbing them to the surface of a microporous structure surface-modified with a positively charged substance; and a tangential flow filtration device for discharging foreign substances through micro-sized pores formed in a hollow fiber membrane formed laterally with respect to the direction in which the extracellular vesicle solution flows.

[0039] It further includes a first pipe connecting the supply container and the microfilter structure; a first pump installed in the first pipe; a second pipe connecting the microfilter structure and the tangential flow filtration device; and a second pump installed in the second pipe.

[0040] It further includes a third pipe connecting the tangential flow filter device and the discharge container; a third pump installed in the third pipe; and a clamp installed at a connection portion between the tangential flow filter device and the third pipe to control opening or closing the third pipe.

[0041] According to the present invention, exosomes or extracellular vesicles can be separated and purified economically and efficiently with a very high purity compared to existing technologies, and separation and purification of extracellular vesicles in large quantities are possible, making them suitable for GMP, and there is an effect of being able to separate and purify exosomes or extracellular vesicles with high purity and uniform particle size distribution economically and efficiently with a high yield.

[0042] Figure 1 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to the first embodiment of the present invention.

[0043] Figure 2 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to a second embodiment of the present invention.

[0044] Figure 3 is a flowchart showing the process of the extracellular vesicle extraction method of the present invention.

[0045] Figure 4 is a schematic diagram showing the separation principle of a fine filter structure.

[0046] Fig. 5 is a cross-sectional view showing the structure of a fine filter structure.

[0047] Fig. 6 is a schematic diagram showing a fine filter structure according to another embodiment.

[0048] Figure 7 is a schematic diagram showing the separation principle of a tangential flow filter device.

[0049] Figure 8 is a graph comparing the sizes of separated particles according to filtration methods.

[0050] Figure 9 is a graph comparing the concentration of separated particles according to filtration method.

[0051] Figure 10 is a graph comparing protein concentration according to filtration method.

[0052] Figure 11 is a table showing the conditions of method 1 of applying a tangential flow filtration device after filtration of a microfilter structure.

[0053] Figure 12 is a table showing the conditions of method 2 of applying a microfilter structure after filtration of a tangential flow filter.

[0054] Figure 13 is a graph comparing protein concentration according to Method 1 and Method 2.

[0055] Figures 14 to 17 are graphs comparing the concentration of particles per unit volume.

[0056] Figure 18 is a graph comparing the concentration of particles per unit volume according to Method 1 and Method 2.

[0057] Figure 19 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to a third embodiment of the present invention.

[0058] Hereinafter, with reference to the drawings, a “method and device for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity” according to an embodiment of the present invention will be described.

[0059] FIG. 1 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to a first embodiment of the present invention, FIG. 2 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to a second embodiment of the present invention, FIG. 3 is a flowchart showing the process of an extracellular vesicle extraction method of the present invention, FIG. 4 is a schematic diagram showing the separation principle of a microfilter structure, FIG. 5 is a cross-sectional diagram showing the structure of a microfilter structure, FIG. 6 is a schematic diagram showing a microfilter structure according to another embodiment, and FIG. 7 is a schematic diagram showing the separation principle of a tangential flow filtration device.

[0060] The present invention is a technology for extracting and purifying extracellular vesicles or exosomes in large quantities from cells or viruses contained in a biological sample, characterized by applying a two-step filtration process and an extracellular vesicle separation process while continuously injecting a large amount of culture solution.

[0061] To this end, a “device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity” (hereinafter referred to as “extraction device”) as shown in FIGS. 1 and 2 is applied to the extraction and purification process.

[0062] The extraction device of the present invention may be composed of a means for supplying an extracellular vesicle solution (a solution in which extracellular vesicles or exosomes are cultured), a means for filtering extracellular vesicles from the extracellular vesicle solution, a means for discharging fine-sized foreign substances contained in the extracellular vesicle solution to the outside, and a means for separating extracellular vesicles from the filter after filtration. In addition, the means for filtering extracellular vesicles may be arranged in a direction perpendicular to the flow direction of the solution, and the means for discharging foreign substances may be arranged in a direction horizontal to the flow direction of the solution.

[0063] The extraction device (100) according to the first embodiment of FIG. 1 and the extraction device (200) according to the second embodiment of FIG. 2 have the same basic structure, but the number of microfilter structures is different.

[0064] The extraction device (100, 200) of the present invention is configured such that a tangential flow filtration device is connected in series between two syringes, an extracellular vesicle solution is injected into one of the two syringes, and then the syringes on both sides are operated alternately so that the extracellular vesicle solution reciprocates inside the tangential flow filtration device in the middle, thereby performing a filtration and purification process.

[0065] Foreign substances discharged from the tangential flow filter are stored in a separate discharge container.

[0066] In addition, a microfilter structure is installed inside one or two syringes so that the extracellular vesicles are filtered during the movement of the solution by the movement of the syringes. Through this structure, the filtration (adsorption) of extracellular vesicles occurs in the syringe, and the tangential flow filtration device discharges foreign substances, thereby increasing the filtration efficiency of the extracellular vesicles and enhancing the purity.

[0067]

[0068] Example 1

[0069]

[0070] The extraction device (100) according to the first embodiment includes a first syringe (102) and a second syringe (104), and the outlets of the first syringe (102) and the second syringe (104) are connected to both ends of a tangential flow filtration device (106). Through this, the solutions contained in the first syringe (102), the second syringe (104), and the tangential flow filtration device (106) can be moved by the operation of the syringes.

[0071] An outlet (110) is formed on one side of the tangential flow filtration device (106), and a discharge container (108) is connected to the outlet (110). Foreign substances contained in the extracellular vesicles are discharged through the outlet (110) and stored in the discharge container (108). The foreign substances are foreign substances such as micro-sized proteins discharged by the tangential flow filtration device (106), and refer to substances other than the extracellular vesicles that are the target of filtration in the present invention.

[0072] A microfilter structure (112) is installed inside the first syringe (102). As illustrated in FIG. 4, a filter made of a membrane or porous particles is installed inside the microfilter structure (112), and charged particles are bound to the surface of the filter. The filtration action is performed when extracellular vesicles are bound to the charged particles by an electric force. The extracellular vesicles bound to the filter are separated by the introduction of a dissolving solution, etc., and the filtration action of the microfilter structure (112) will be described later.

[0073] The second syringe (104) is not equipped with a separate filtering device or separation device, and is configured to perform the function of applying pressure for movement of the extracellular vesicle solution.

[0074] The separation process is carried out while the extracellular vesicle solution containing the extracellular vesicles to be separated is stored in the first syringe (102). Initially, the second syringe (104) is empty and the piston is inserted into the cylinder.

[0075] First, an extracellular vesicle solution is prepared and injected into the first syringe (102). (S102) A preliminary filtration process using a preliminary filter may be added to remove impurities contained in the extracellular vesicle solution injected into the first syringe (102).

[0076] Then, the piston of the first syringe (102) is pressed to discharge the extracellular vesicle solution, and the piston of the second syringe (104) is pulled out to allow the extracellular vesicle solution to flow in. In some cases, it may be configured so that only the piston of the first syringe (102) that discharges the extracellular vesicle solution is pressed. In this way, the solution can move due to the positive pressure generated in the first syringe (102) that discharges the extracellular vesicle solution and the negative pressure generated in the second syringe (104) that introduces the extracellular vesicle solution.

[0077] The extracellular vesicle solution, in which the extracellular vesicles are not separated, exits the outlet of the first syringe (102). In this process, the extracellular vesicles are adsorbed to the microfilter structure (112) mounted inside the first syringe (102) and are first filtered (S104).

[0078] And the extracellular vesicle solution flows into one side of the tangential flow filtration device (106) and moves to the other side, where fine-sized substances are discharged to the side, thereby performing secondary filtration (S106).

[0079] The tangential flow filtration device (106) has a structure in which a hollow fiber membrane is formed in a horizontal direction relative to the direction in which the extracellular vesicle solution flows, as illustrated in FIG. 7. In some cases, the device may be configured to move in a direction parallel to the direction in which the solution moves, or in a direction that is separated by an angle less than 90°.

[0080] The tangential flow filtration device (106) has a longitudinal path for the extracellular vesicle solution to move, and an outlet section made of a hollow fiber membrane is formed on one side of the path. Micro-sized pores are formed in the hollow fiber membrane.

[0081] The size of the pores formed in the hollow fiber membrane may be 100 nm or less, 80 nm or less, 60 nm or less, or 40 nm or less. However, considering that the size of typical extracellular vesicles is 50 nm or more, the pore size is preferably 50 nm or less. The pore size of hollow fiber membranes readily available on the market is 10 nm to 40 nm.

[0082] Using a tangential flow filtration device (106) having pores of this size, foreign substances such as particles having a size of 50 nm or less or proteins having a molecular weight of 100 kDa or 500 kDa or less can be removed. In the present invention, cell debris, proteins, and other particles separated and discharged by the tangential flow filtration device (106) are defined as "foreign substances."

[0083] In addition, the hollow fiber membrane used in the tangential flow filtration device (106) may be a conventional filtration membrane, mesh, fine particle layer, or porous material in addition to a hollow fiber.

[0084] When the microfilter structure (112) and the tangential flow filtration device (106) are connected in series as in the present invention, the microfilter structure (112) captures only nano-sized particles having a negative charge, and foreign substances of 50 nm or less can be removed through the tangential flow filtration device (106).

[0085] The extracellular vesicle solution, which has undergone primary and secondary filtration while passing through the tangential flow filtration device (106), flows into the second syringe (104). At this time, the piston of the second syringe (104) is pushed out of the cylinder, allowing the solution to enter inside. Foreign substances discharged through the hollow fiber membrane of the tangential flow filtration device (106) are stored in the discharge container (108) through the discharge port (110) formed on one side.

[0086] In this state, when the piston of the second syringe (104) is pressed to cause the extracellular vesicle solution to move in the opposite direction, the solution passes through the tangential flow filtration device (106) and foreign substances are discharged, and as it enters the interior of the first syringe (104), the extracellular vesicles are repeatedly filtered by the microfilter structure (112). (S108) This first filtration and second filtration are repeated while changing the direction of movement of the solution.

[0087] When the filtration is completed through the set number of repetitions, the first syringe (104) is separated, and the eluent is injected into the first syringe (104) to proceed with the separation process (S110). In some cases, a method of separating the microfilter structure (112) from the first syringe (104) and injecting the eluent into the separated microfilter structure (112) may also be applied.

[0088] As the eluent is injected, the negatively charged nanoparticles attached to the microfilter structure (112) are detached from the filter surface, thereby separating the extracellular vesicles (S112). When a hybrid process is applied in which the two-stage separation means of the microfilter structure (112) and the tangential flow filtration device (106) are connected in series, it is possible to separate and purify only the negatively charged extracellular vesicles or proteins of 50 nm or more in size.

[0089] As illustrated in FIG. 4, the microfilter structure (112) is formed as a microporous structure in which a plurality of pores are formed, and filtration is performed in such a way that the extracellular vesicles remain in the microporous structure as the extracellular vesicle solution passes through the microporous structure. The surface of the microporous structure is manufactured in a form in which a material having a specific charge is attached, and the extracellular vesicles are bound to the surface of the filter material by the charge. Typically, extracellular vesicles or exosomes included in the extracellular vesicle solution have negative charge characteristics, so it is preferable to modify the material surface of the microporous structure inside the microfilter structure (112) with a polycationic material having positive charge characteristics.

[0090] The polycationic material used for surface modification of the microfilter structure (112) is any one of polylysine, protamine, polyarginine, polyhistidine, cationic dextran, cationic dendrimer, cationic polysaccharide, polyamidoamine, polyethyleneimine, polyquaternium, poly-2-dimethylaminoethyl methacrylate (PDMAEMA), poly-2-dimethylaminomethyl styrene (PDMAMS), poly-1-vinylpyrrolidone (p1-VP), poly diethylaminoethyl acrylate (pDEAEA), poly dimethylaminoethyl acrylate (pDMAEA), poly diethylaminoethyl methacrylate (pDEAMA), lipopolyamines, quaternary ammoniums, guanidine, imidazole, polyaniline, polypyrrol, and chitosan. It could be strange.

[0091] The microporous structure applied to the microfilter structure (112) can be formed into any one of a microparticle structure including a stacked structure of spherical beads, a membrane structure, and a mesh structure.

[0092] In the case of a microparticle structure, pores are formed within the layered structure so that the extracellular vesicle solution passes through them, allowing the extracellular vesicles contained within to bind electrically to the cationic surface of the microparticles. The cationic layer attached to the surface of the microparticles attracts and binds the negatively charged extracellular vesicles through electrical attraction. Therefore, the size of the pores between the microparticles need not be smaller than the size of the extracellular vesicles. Rather, it is preferable that they be large enough to allow the smooth passage of the extracellular vesicle solution.

[0093] The size of the microparticles and pores may vary depending on the material, but for spherical microparticles, the ratio of the diameter of the extracellular vesicles to the microparticles is preferably 1:5 to 1:10. When microparticles of this size are used, the size of the pores formed inside may be about 2 to 5 times the diameter of the extracellular vesicles, and the pore size is preferably configured to be 800 nm or larger.

[0094] The microparticles used in the present invention may be at least one of polyacrylate, polyacrylamide, polymethacrylate, polyethylene glycol, polystyrene vinyl benzene, polystyrene, hydrogel, agarose, ceramic, silica, latex, metal particles, glass particles, and magnetic particles.

[0095] The microfilter structure (112) may be formed as a membrane structure or a mesh structure, in which case a material capable of adsorbing a polyvalent cation substance is used. As a material for the membrane or mesh, any one or more of nylon, polyethylene, polypropylene, polyethylene terephthalate (PET), polylactic acid (PLA), polyethersulfone (PES), cellulose acetate (CA), polyvinylidene fluoride (PVDF), and polycarbonate (PC) may be used. Similar to the above-described microparticle structure, the membrane or mesh is also manufactured with a polyvalent cation substance adsorbed on the surface.

[0096] The membrane or mesh structure may be single-layered, or in some cases, multi-layered to increase filtration efficiency. In the case of a multi-layer configuration, it is preferable to form a structure in which 10 or more single layers are laminated.

[0097] A dissolving solution is injected to separate the extracellular vesicles adsorbed to the microfilter structure (112).

[0098] The elution process using an eluent can be a single process, but can also be configured as multiple processes to increase elution efficiency. In the present invention, elution efficiency can be improved by sequentially introducing two types of eluents.

[0099] The first is configured to perform a first washing of the microfilter structure (112) using the first reagent under the first condition. The first reagent is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH -A material containing one or more of the above may be used. And, the first condition may be set to be performed at least once within a pH range of one of pH 5.5 to 6.5 and pH 7.5 to 9.5.

[0100] The second is configured to perform a second washing of the microfilter structure (112) under the second condition using the second reagent. The second reagent is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH - A substance containing one or more of the above may be used, and it is preferable to use it at a concentration of 0.2 M to 3 M. In addition, the second condition may be set to be performed at least once while mixing at 500 rpm to 2000 rpm for a time of 1 minute to 60 minutes.

[0101] The elution process can be carried out in a step of introducing the first reagent under the first condition and a step of introducing the second reagent under the second condition.

[0102] After the elution process is completed under the first and second conditions, an additional buffer replacement step can be added, and the buffer replaced in this step can be one or more of physiological saline solution, deionized water, and distilled water.

[0103] The extraction and purification process of the extracellular vesicles is completed by processing the separation solution of the first syringe (102) in which the extracellular vesicles are stored in a separated state through a separate process.

[0104]

[0105] Second Example

[0106]

[0107] The extraction device (200) according to the second embodiment has the same basic configuration as the extraction device (100) according to the first embodiment. That is, it includes a first syringe (202) and a second syringe (204), and the outlets of the first syringe (202) and the second syringe (204) are connected to both ends of a tangential flow filtration device (206). Through this, the solutions contained in the first syringe (202), the second syringe (204), and the tangential flow filtration device (206) can be moved by the operation of the syringes.

[0108] Additionally, foreign substances separated by the tangential flow filter (206) are stored in the discharge container (208) through the discharge port (210).

[0109] In the second embodiment, a first microfilter structure (212) is installed inside the first syringe (202), and a second microfilter structure (214) is installed in the second syringe (204). That is, by installing microfilter structures in both syringes, separation efficiency can be improved.

[0110] In this state, the extracellular vesicle solution is injected into either the first syringe (202) or the second syringe (204), or into both the first syringe (202) and the second syringe (204), and the process of extracellular vesicle filtration and foreign substance separation is performed while moving the solution to the left and right.

[0111] After the repeated filtration is completed, the first syringe (202) and the second syringe (204) can be separated and the extracellular vesicles can be separated by injecting the eluent. As in the first embodiment, the first microfilter structure (212) and the second microfilter structure (214) can be separated from the syringe and the elution process can be performed in a separate device.

[0112]

[0113] Meanwhile, Fig. 5 is a cross-sectional view showing the structure of a fine filter structure, and Fig. 6 is a schematic diagram showing a fine filter structure according to another embodiment.

[0114] As described above, when the filter membrane (61) is formed of a microparticle (63) structure, the size of the pores (64) between the microparticles (63) is formed to be relatively larger than the size of the extracellular vesicles (EV). The microparticles (63) are laminated between the filter membrane (61) and the side wall (62). In addition, the filter membrane (61) can be formed in multiple layers.

[0115] Extracellular vesicles are charged by cationic substances adsorbed on the surface of microparticles (63). Since the size of the extracellular vesicles is relatively smaller than the size of the pores (64), the extracellular vesicle solution can pass through smoothly even when the extracellular vesicles are sufficiently adsorbed.

[0116] Even in the case of a membrane structure or mesh structure, the size of the pores formed by the lattice-shaped yarn (65) is still considerably larger than the size of the extracellular vesicles, so that the solution of the extracellular vesicles can move smoothly even when the extracellular vesicles are sufficiently adsorbed to the yarn (65).

[0117] Fig. 8 is a graph comparing the sizes of separated particles according to filtration methods, Fig. 9 is a graph comparing the concentration of separated particles according to filtration methods, and Fig. 10 is a graph comparing the concentration of protein according to filtration methods.

[0118] As shown in Fig. 8, in the results of the comparative example (UC) in which the microfilter structure (ExoFilter) or tangential flow filtration device (TFF) was not applied, the particle size was relatively the largest, and when the tangential flow filtration device and microfilter structure were applied, it was confirmed that the size of the contained particles was sequentially smaller.

[0119] In addition, as shown in Fig. 9, it can be confirmed that the concentration of the separated extracellular vesicles is the greatest when a microfilter structure is applied, and is relatively small when a tangential flow filtration device is applied.

[0120] Conversely, as shown in Fig. 10, the concentration of protein particles contained within the filtered solution is highest in the comparative example, followed by the tangential flow filtration device and the microfilter structure, in that order.

[0121] These results confirm that the filtration efficiency is highest when the microfilter structure is applied alone, followed by when the tangential flow filtration device is applied.

[0122] Meanwhile, Fig. 11 is a table showing the conditions of method 1 for applying a tangential flow filtration device after filtration of a microfilter structure, Fig. 12 is a table showing the conditions of method 2 for applying a microfilter structure after filtration of a tangential flow filtration device, Fig. 13 is a graph comparing protein concentrations according to methods 1 and 2, Figs. 14 to 17 are graphs comparing the concentrations of particles per unit volume, and Fig. 18 is a graph comparing the concentrations of particles per unit volume according to methods 1 and 2.

[0123] The microfilter structure and tangential flow filtration device used in the present invention can separate extracellular vesicles in different ways. The microfilter structure separates extracellular vesicles by adsorbing them through charge binding, while the tangential flow filtration device separates extracellular vesicles by removing relatively small foreign substances. A key feature of the present invention lies in the ability to achieve greater effectiveness by combining these two filtration devices.

[0124] However, when two filter devices are simply connected in series, it was confirmed that there is a difference in separation efficiency depending on which method is applied first.

[0125] Fig. 11 describes the filtration conditions according to Method 1. In Method 1, the extracellular vesicle solution is first passed through a microfilter structure to perform primary filtration, and the primary filtered solution is then passed through a tangential flow filtration device to perform secondary filtration.

[0126] And the filtration conditions by method 2 described in Fig. 12 applied a method of performing primary filtration in a tangential flow filtration device and secondary filtration in a microfilter structure.

[0127] The specifications and materials of the microfilter structure and tangential flow filtration device used in the two experiments, as well as the concentration of the sample solution, were all the same, and only the filtration order of the two filtration devices was changed for the experiments.

[0128] The results shown in Figure 13 show the concentration of protein particles when the tangential flow filter (TFF) and the microfilter structure (ExoFilter) were used alone, when Method 1 (ExoFilter+TFF) was applied, and when Method 2 (TFF+ExoFilter) was applied.

[0129] It can be confirmed that the concentrations when the tangential flow filter (TFF) and the microfilter structure (ExoFilter) were used alone were 411 and 516, respectively, the concentration was 53 when method 1 was applied, and the concentration was 228 when method 2 was applied.

[0130] Therefore, when two filtration devices are applied sequentially, efficiency is significantly improved compared to using each device individually. Furthermore, when connecting and using two filtration devices, it can be seen that performing secondary filtration using a tangential flow filtration device after primary filtration using a microfilter structure is relatively more efficient.

[0131] Through the graphs of Figures 14 to 18, it can be confirmed that the results of the four cases of filtering appear in the same order.

[0132]

[0133] Third Example

[0134]

[0135] Meanwhile, Fig. 19 is a conceptual diagram showing the structure of an extracellular vesicle extraction device according to the third embodiment of the present invention.

[0136] The extraction device (300) according to the third embodiment is characterized by being configured as an automated device by using a pump to provide pressure for movement of an extracellular vesicle solution passing through a microfilter structure and a tangential flow filtration device.

[0137] The supply container (302) stores an extracellular vesicle solution including extracellular vesicles to be separated, and supplies the stored solution to a filter and a filtration device.

[0138] The microfilter structure (306) filters extracellular vesicles by adsorbing them through a charge binding method, using the same principle as described above.

[0139] The tangential flow filtration device (310) separates and discharges foreign substances such as proteins contained in the extracellular vesicle solution that has passed through the microfilter structure (306).

[0140] Foreign substances separated in the tangential flow filter (310) are stored in the discharge container (316).

[0141] The supply container (302) and the microfilter structure (306) are connected by a first pipe (318), and a first pump (304) is installed in the first pipe (318). The extracellular vesicle solution before being filtered flows through the first pipe (318) by the pressure generated by the first pump (304).

[0142] The microfilter structure (306) and the tangential flow filtration device (310) are connected by a second pipe (320), and a second pump (308) is installed in the second pipe (320). A solution in which extracellular vesicles are separated by the microfilter structure (306) and foreign substances are separated by the tangential flow filtration device (310) flows through the second pipe (320) by the pressure generated by the second pump (308).

[0143] The tangential flow filter (310) and the discharge container (316) are connected by a third pipe (322), and a third pump (314) is installed in the third pipe (322). A solution containing foreign substances separated by the tangential flow filter (310) flows through the third pipe (322) by the pressure generated by the third pump (314).

[0144] A clamp (312) may be added to the connection between the tangential flow filter (310) and the third pipe (322). The clamp (312) has a function of opening or closing the third pipe (322), and is controlled to be closed or opened depending on the operating status of the extraction device (300).

[0145] In an extraction device (300) having such a configuration, extracellular vesicles are separated in the same manner as described in the first and second embodiments.

[0146] The extracellular vesicle solution stored in the supply container (302) is introduced into the microfilter structure (306) along the first pipe (318). The microfilter structure (306) filters the extracellular vesicles by adsorbing them onto the surface of a membrane charged with cations.

[0147] The extracellular vesicle solution that has passed through the microfilter structure (306) is introduced into a tangential flow filtration device (310), and foreign substances are discharged through the pores of the hollow fiber membrane arranged in a direction parallel to the direction of movement. The discharged foreign substances are stored in the discharge container (316) through the third pipe (322).

[0148] The extracellular vesicle solution that has passed through the tangential flow filtration device (310) is circulated through the second pipe (320) and then injected into the microfilter structure (306) again, and filtration and separation are performed while repeatedly passing through the microfilter structure (306) and the tangential flow filtration device (310).

[0149] The control unit (not shown in the drawing) stops the pump operation after filtration is completed by circulating the pump for a set number of times, and the operator proceeds with the process of separating extracellular vesicles by separating the microfilter structure (306) and injecting the eluent.

[0150] Although the preferred embodiments of the present invention have been described with reference to the attached drawings, it will be understood by those skilled in the art that the technical configuration of the present invention described above can be implemented in other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A method for extracting extracellular vesicles from an extracellular vesicle solution, A first step of preliminarily filtering the above extracellular vesicle solution; A second step of adsorbing the extracellular vesicles by introducing the filtered extracellular vesicle solution into a microfilter structure composed of a microporous structure whose surface is modified with a positively charged substance; A third step of removing foreign substances below a certain size by putting the sample liquid that has passed through the second step into a tangential flow filtration device; A fourth step of repeating the second and third steps a predetermined number of times; A method for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, comprising a fifth step of injecting a dissolving solution into the microfilter structure to separate extracellular vesicles.

2. In paragraph 1, A method for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, characterized in that the above microfilter structure is formed of a microporous structure of any one of microparticles, a membrane structure, a mesh structure, and a stacked structure of spherical beads.

3. In paragraph 1, A method for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the microfilter structure is installed inside a syringe storing the extracellular vesicle solution.

4. In paragraph 1, A method for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, characterized in that the surface of the above microporous structure is modified by a polycationic substance.

5. In paragraph 4, The above polyvalent cationic material is characterized by being at least one of polylysine, protamine, polyarginine, polyhistidine, cationic dextran, cationic dendrimer, cationic polysaccharide, polyamidoamine, polyethyleneimine, polyquaternium, poly-2-dimethylaminoethyl methacrylate (PDMAEMA), poly-2-dimethylaminomethyl styrene (PDMAMS), poly-1-vinylpyrrolidone (p1-VP), poly diethylaminoethyl acrylate (pDEAEA), poly dimethylaminoethyl acrylate (pDMAEA), poly diethylaminoethyl methacrylate (pDEAMA), lipopolyamines, quaternary ammoniums, guanidine, imidazole, polyaniline, polypyrrol, and chitosan, and extracting extracellular vesicles in large quantities from a biological sample. A method for purifying to high purity.

6. In paragraph 2, The microparticles contained in the above microporous structure are A method for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the extracellular vesicles are composed of at least one of polyacrylate, polyacrylamide, polymethacrylate, polyethylene glycol, polystyrene vinyl benzene, polystyrene, hydrogel, agarose, ceramic, silica, latex, metal particles, glass particles, and magnetic particles.

7. In paragraph 1, The fifth step of separating the extracellular vesicles by injecting the eluent includes the fifth-first step of injecting the first reagent into the microfilter structure and washing the microfilter structure under the first condition. The first reagent above is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH - A substance containing one or more of the following: A method for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, characterized in that the first condition is set to be performed at least once within a pH range of any one of pH 5.5 to 6.5 and pH 7.5 to 9.

5.

8. In paragraph 7, After the above step 5-1, a second reagent is added to the microfilter structure, and a step 5-2 is further included to wash the microfilter structure under the second condition. The second reagent is Cl - , CH3COO - , SO4 2- , HCO - , SiO - and OH - A substance having a concentration of 0.2 M to 3 M, which contains at least one of: A method for extracting a large amount of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the second condition is set to be performed at least once while mixing at 500 rpm to 2000 rpm for a period of 1 to 60 minutes.

9. In paragraph 8, Further comprising a step 5-3 that performs additional buffer substitution after the step 5-2 above, A method for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, characterized in that the buffer substituted in the above step 5-3 is at least one of physiological saline solution, deionized water, and distilled water.

10. A device for extracting extracellular vesicles from an extracellular vesicle solution, Syringe 1; Second syringe and; A tangential flow filter device having both ends connected to the outlets of the first syringe and the second syringe, respectively; An outlet formed on one side of the above tangential flow filter device; A discharge container connected to the above discharge port; A device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, the device comprising a microfilter structure installed inside at least one of the first syringe or the second syringe.

11. In paragraph 10, A device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the extracellular vesicle solution injected into the first syringe passes through the tangential flow filtration device and then flows into the second syringe.

12. In paragraph 11, A device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the extracellular vesicle solution moves by positive pressure inside the first syringe or negative pressure inside the second syringe.

13. In paragraph 10, The above microfilter structure is a device for extracting extracellular vesicles in large quantities from a biological sample and purifying them with high purity, characterized in that the extracellular vesicles contained in the extracellular vesicle solution are filtered by adsorbing them on the surface of a microporous structure surface-modified with a positively charged substance.

14. In paragraph 10, The above tangential flow filtration device has a structure in which a hollow fiber membrane is formed in a direction that is horizontal to the direction in which the extracellular vesicle solution flows or is spread at an angle less than 90°, and is characterized in that foreign substances are discharged through the micro-sized pores formed in the hollow fiber membrane, and is a device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity.

15. In paragraph 14, A device for extracting a large amount of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the size of the pores formed in the hollow fiber membrane is 50 nm or less.

16. In paragraph 13, A device for extracting a large amount of extracellular vesicles from a biological sample and purifying them with high purity, characterized in that the size of the pores formed inside the microporous structure is 800 nm or more.

17. A device for extracting extracellular vesicles from an extracellular vesicle solution, A supply container for storing an extracellular vesicle solution including extracellular vesicles to be separated, and supplying the stored solution; A microfilter structure that filters extracellular vesicles contained in the above extracellular vesicle solution by adsorbing them on the surface of a microporous structure surface-modified with a positively charged substance; A device for extracting a large quantity of extracellular vesicles from a biological sample and purifying them with high purity, comprising a tangential flow filtration device that discharges foreign substances through micro-sized pores formed in a hollow fiber membrane formed laterally with respect to the direction in which the extracellular vesicle solution flows.

18. In paragraph 17, A first pipe connecting the supply container and the microfilter structure; A first pump installed in the first pipe; A second pipe connecting the above microfilter structure and the above tangential flow filtration device; A device for extracting a large amount of extracellular vesicles from a biological sample and purifying them with high purity, further comprising a second pump installed in the second pipe.

19. In paragraph 18, A third pipe connecting the above tangential flow filter device and the discharge container; A third pump installed in the third pipe; A device for extracting a large amount of extracellular vesicles from a biological sample and purifying them with high purity, further comprising a clamp installed at a connection portion between the tangential flow filter device and the third pipe and controlling the opening or closing of the third pipe.

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