Method for producing extracellular vesicles

Abstract

The purpose of the present invention is to provide a purification method by which extracellular vesicles can be separated and purified at a high recovery rate and at low cost. The present invention pertains to a method for producing extracellular vesicles, the method comprising: a step (a) for obtaining a biological sample containing extracellular vesicles; a step (b) for adjusting the electrical conductivity of the biological sample to 4-15 mS / cm; a step (c) for bringing the biological sample into contact with a carrier having a positive charge; and a step (d) for bringing a metal salt-containing buffer solution into contact with the carrier to recover extracellular vesicles.

Description

Method for producing extracellular vesicles

[0001] The present invention relates to a method for producing extracellular vesicles by purification with high recovery rate.

[0002] Extracellular vesicles (EVVs) are a collective term for vesicles with heterogeneous lipid bilayer structures secreted by almost all living cells. Based on differences in their production mechanisms, they are broadly classified into exosomes, microvesicles, and apoptotic bodies.

[0003] Exosomes are formed by the inward budding of late endosomal membranes, which then fuse with the plasma membrane to form complete particles that are then secreted extracellularly via exocytosis. Exosomes are extracellular vesicles with a diameter of approximately 30 to 150 nm that are released by eukaryotic cells, including animals, plants, and fungi. They are known to play an important role in intercellular signaling, such as encapsulating and propagating proteins, mRNA, miRNA, and other molecules in body fluids. In recent years, it has been suggested that exosomes are involved in the promotion of cancer metastasis and its aggravation. Diagnostic markers focusing on exosomes have been developed, including biomarkers for predicting cancer progression, metastasis, and prognosis, using miRNAs expressed from cancer-derived exosomes as tumor markers.

[0004] Furthermore, by isolating and purifying exosomes from living organisms, they are expected to be applied to drug delivery systems (DDS) as biologically derived transporters encapsulating intracellular nucleic acids, proteins, etc. Therefore, there is a demand for technology to isolate and purify exosomes with high purity.

[0005] Microvesicles are produced by the outward budding and separation of cell membranes. Microvesicles range in size from 100 to 1,000 nm in diameter. Apoptotic bodies are produced when cells undergo apoptosis and range in size from 50 to 5,000 nm in diameter. Microvesicles and apoptotic bodies are expected to have similar applications to exosomes.

[0006] Methods for separating and purifying extracellular vesicles include centrifugal fractionation, ultracentrifugation such as density gradient centrifugation, precipitation using polymers such as polyethylene glycol, and chromatography such as size exclusion chromatography and affinity chromatography. Ultracentrifugation is a common method, and highly purified extracellular vesicles can sometimes be obtained by density gradient centrifugation. For example, Patent Document 1 discloses a method for producing exosomes in which a biological sample is ultrafiltered and then subjected to anion exchange column chromatography.

[0007] International Publication No. 2020 / 027185 Pamphlet

[0008] Ultracentrifugation has problems such as structural changes to extracellular vesicles during the centrifugation process, a decrease in quality due to destruction, and a low recovery rate. In addition, methods using anion exchange column chromatography have the problem of an insufficient number of extracellular vesicles adsorbed per unit volume.

[0009] The present invention aims to provide a purification method that can separate and purify extracellular vesicles with a high recovery rate and at low cost, and a method for producing extracellular vesicles using the purification method.

[0010] As a result of extensive research into methods for preparing extracellular vesicles, the inventors have found that by optimizing the series of steps from cell culture, removal of contaminants, and separation and recovery of extracellular vesicles, it is possible to recover the desired extracellular vesicles with high purity and at a high recovery rate.

[0011] That is, the present inventors have found that the above-mentioned problems can be solved by the following means, and have arrived at the present invention. [1] A method for producing extracellular vesicles, comprising: step (a) obtaining a biological sample containing extracellular vesicles; step (b) adjusting the electrical conductivity of the biological sample to 4 mS / cm or more and 15 mS / cm or less; step (c) contacting the biological sample with a positively charged carrier; and step (d) contacting the carrier with a buffer containing a metal salt to recover extracellular vesicles. [2] The method according to [1], wherein step (a) comprises isolating cells from an animal or thawing frozen cells from a cell stock, followed by culturing. [3] The method according to [1] or [2], wherein step (a) and step (b) comprise a step of removing at least a portion of contaminants from the biological sample. [4] The method according to [3], wherein the step of removing at least a portion of contaminants is one or more methods selected from the group consisting of centrifugation, microfiltration, ultrafiltration, and dialysis. [5] The method according to any one of [1] to [4], wherein in step (c), the carrier is a separation membrane having anion exchange capacity. [6] The method according to any one of [1] to [5], wherein in step (c), the carrier has a tertiary amine and / or a quaternary ammonium group. [7] The method according to any one of [1] to [6], wherein, between step (c) and step (d), a step of contacting the carrier with a buffer solution to wash the carrier is included. [8] The method according to any one of [1] to [7], wherein in step (d), the buffer solution containing a metal salt is any one of Tris-HCl buffer, phosphate-buffered saline, and Tris-buffered saline. [9] The method according to any one of [1] to [8], wherein in step (d), the metal salt is NaCl.

[10] The method according to any one of [1] to [9], wherein in step (d), the electrical conductivity of the sample containing recovered extracellular vesicles is adjusted to more than 15 mS / cm.

[11] The method according to any one of [1] to

[10] , wherein the extracellular vesicles are exosomes and / or microvesicles.

[0012] In the method for purifying extracellular vesicles from a biological sample, the present invention includes a step of adjusting the electrical conductivity of the biological sample before contacting the biological sample with a positively charged carrier, thereby maximizing the amount of target extracellular vesicles adsorbed onto the carrier. Furthermore, it is possible to obtain a particle group in which an extracellular vesicle marker is strongly expressed per particle.

[0013] 1 is a schematic diagram showing an example of a culture device for preparing a biological sample containing extracellular vesicles. 2 is a graph (calibration curve) showing the relationship between salt concentration and electrical conductivity. 3 is a photograph showing the results of Western blotting.

[0014] The present invention relates to a novel method for purifying and producing extracellular vesicles, including exosomes, from biological samples. The present invention is described below, but is not limited to the following specific examples. In this disclosure, the numerical range "x to y" includes both x and y.

[0015] 1. Biological sample preparation step In this step, a biological sample containing the target extracellular vesicles is prepared.

[0016] The biological sample is not particularly limited as long as it contains extracellular vesicles to be purified or produced, but is preferably a liquid. Specifically, it can be a cell culture medium, a culture supernatant separated from a cell culture medium, interstitial fluid, blood, serum or plasma separated from blood, urine, cerebrospinal fluid, lymph, ascites, milk, etc. If the biological sample is not a liquid, it is preferably dissolved or dispersed in a liquid such as a buffer solution.

[0017] In the present invention, the method for purifying extracellular vesicles from a biological sample preferably includes the steps of obtaining a biological sample containing extracellular vesicles, adjusting the electrical conductivity of the biological sample, contacting the biological sample with a positively charged carrier, contacting the carrier with a buffer solution and washing it, and contacting the carrier with a buffer solution containing a metal salt and recovering the extracellular vesicles.

[0018] Extracellular vesicles (EVs) are a collective term for vesicles with heterogeneous lipid bilayer structures secreted by almost all living cells. Extracellular vesicles are broadly divided into three types: exosomes, microvesicles, and apoptotic bodies, based on differences in their intracellular production mechanisms. Exosomes are 30-150 nm in diameter. Exosomes are formed by inward budding of late endosomal membranes, which then fuse with the plasma membrane to form complete particles that are secreted extracellularly via exocytosis. Microvesicles, on the other hand, are generated by outward budding and separation of the plasma membrane. They are 100-1,000 nm in diameter. Apoptotic bodies are generated when cells undergo organized cell death (apoptosis). They are 50-5,000 nm in diameter.

[0019] In the present invention, "extracellular vesicles" may be extracellular vesicles contained in a biological sample or extracellular vesicles produced by the target cultured cells, and there are no particular limitations on the size or shape of the extracellular vesicles themselves. Furthermore, they may be natural or non-natural (artificial) extracellular vesicles.

[0020] Extracellular vesicles can be prepared, for example, from cultured cells. In the present invention, "cultured cells" generally refers to cells cultured in vitro (outside the body) and is not limited to specific commercially established autologous lines (cell lines). When cells, tissues, organs, etc. extracted from human or non-human organisms, particularly mammalian living bodies, are cultured in vitro, cells contained in the culture (e.g., primary culture) are also included in "cultured cells." Cells constituting various commercially established cell lines, such as cultured tumor cells, are typical examples of "cultured cells" as used herein. Note that "tumor cells," such as "cancer cells," are cells that form various tumors, typically cells that have reached the stage of abnormal proliferation independent of surrounding normal tissues, including so-called cancerous cells. "Cultured tumor cells" derived from cells constituting various cell types, such as lymphomas and melanomas, are suitable examples of "cultured cells" as used herein. Examples of cultured tumor cells include cultured neuroblasts and retinoblasts.

[0021] The cell culture includes the target cultured cells and materials constituting the culture medium. The cultured cells include commonly available, typically commercially available cell lines, such as cultured tumor cell lines derived from humans or non-human mammals. For example, cultured tumor cells such as uterine cancer, ovarian cancer, and breast cancer cells can be used, and HEK293 cells derived from human fetal kidney can be used.

[0022] The materials constituting the culture medium for cultured cells may be in accordance with conventionally recommended medium compositions depending on the type of cultured cells used, and there are no particular limitations on the implementation of the present invention. For example, a medium prepared by adding serum such as fetal bovine serum (FBS) and, if necessary, components such as antibiotics to commercially available media such as α-MEM, DMEM, or MEM can be used. Here, from the perspective of recovering highly pure extracellular vesicles, it is preferable to use a serum-containing medium that is not contaminated with serum-derived animal extracellular vesicles.

[0023] In the present invention, when extracellular vesicles are isolated from the culture supernatant, the cells to be cultured can be obtained by thawing frozen cells from a cell stock. In another embodiment, the cells can be obtained by isolation from animal tissue, including human tissue. Examples of the cells used include cells contained in blood, stem cells, etc. The medium used for culture may be either serum-free or serum-containing. Examples of the cells contained in blood include T cells. Examples of stem cells include ES cells, iPS cells, and mesenchymal stem cells.

[0024] The culture method is not particularly limited, but examples include flat culture using a culture flask, dish, plate, etc.; microcarrier culture in which bead-shaped microcarriers to which cells are attached are suspended in a culture medium and cultured; and culture using hollow fiber membranes or hollow gels.

[0025] The culture period is not particularly limited, but from the viewpoint of efficiently recovering a sufficient amount of extracellular vesicles, a culture period of about 2 to 5 days in a CO2-rich atmosphere at a temperature of about 37° C. The CO2 concentration can be adjusted to, for example, about 5±2%.

[0026] Figure 1 shows one example of the configuration of a hollow fiber membrane-type cell culture device. In Figure 1, 1 and 2 are gas-permeable liquid medium storage containers (e.g., culture bags). A flow path is connected from liquid medium storage container 1 to cell culture vessel 17 via sterile connection connector 13 and valve 4, allowing the medium contained in liquid medium storage container 1 to be transferred to the hollow portion of the hollow fiber membrane. On the other hand, a flow path is connected from liquid medium storage container 2 to cell culture vessel 17 via sterile connection connector 14 and valve 5, allowing the medium contained in liquid medium storage container 2 to be transferred to the outside of the hollow fiber membrane.

[0027] A flow path is connected from the hollow portion of the cell culture vessel 17 via valve 7, valve 11, liquid feed pump 20, valve 12, and sterile connection connector 16 to a waste liquid collection container 18, allowing the culture medium that has passed through the cell culture vessel 17 to be discarded. A liquid feed pump 20 provided in the flow path can control the flow rate and other parameters of supplying a liquid (such as a culture medium) from the liquid culture medium storage container 1 to the cell culture vessel 17, discharging the liquid from the cell culture vessel, and discarding the liquid. On the other hand, a flow path is connected from the outer cavity side of the cell culture vessel 17 via valve 6, valve 9, liquid feed pump 19, valve 10, and sterile connection connector 16 to a waste liquid collection container 18, allowing the culture medium that has passed through the cell culture vessel 17 to be discarded. A liquid feed pump 19 provided in the flow path can control the flow rate and other parameters of supplying a liquid (such as a culture medium) from the liquid culture medium storage container 2 to the cell culture vessel 17, discharging the liquid from the cell culture vessel, and discarding the liquid.

[0028] 1, the cell collection container 3 is in communication with the hollow portion of the cell culture container 17 via valves 7 and 8 and a sterile connection connector 15. If necessary, a pump or the like may be further provided in this flow path to collect cells. Note that cell collection may be performed after removing the cell culture container 17 from the cell culture device. In this case, the configuration from the branch of the waste flow path on the lumen side to the cell collection container 3 may not be necessary.

[0029] In FIG. 1, it is preferable that the CO2 incubator 31 covers most of the above-mentioned components, but as long as at least the gas-permeable liquid culture medium storage containers 1 and 2 and the cell culture container 17 are covered, the other parts may be installed outside the CO2 incubator 31.

[0030] In FIG. 1, an operation controller 21 (operation panel) may be installed to perform the above operations and steps and to perform monitoring.

[0031] In the present invention, the step of obtaining a biological sample containing extracellular vesicles may include a step of removing contaminants such as unnecessary large cellular debris. Examples of steps for removing contaminants include centrifugation, microfiltration, ultrafiltration, and dialysis. Centrifugation is performed, for example, at 4°C, 10,000 x g, for 15 to 30 minutes. The supernatant from which cellular debris and other particles have been removed by centrifugation is recovered. Additional centrifugation may be performed to further remove contaminants. When additional centrifugation is performed, the centrifugal acceleration (g) may be increased to remove medium-sized or smaller cellular debris. When the target of purification is exosomes or microvesicles, apoptotic vesicles may also be removed.

[0032] Microfiltration is a process using a membrane filter with a pore size of 0.2 to 0.5 μm, and by recovering the filtrate, medium-sized cell debris and the like can be removed in the same way as with centrifugation.

[0033] Furthermore, the supernatant obtained by centrifugation and / or the filtrate obtained by microfiltration may be treated by ultrafiltration and / or dialysis using a membrane filter with a pore size of 0.1 μm or less, more preferably 0.05 μm or less. Ultrafiltration and / or dialysis can remove contaminants smaller than extracellular vesicles and concentrate extracellular vesicles.

[0034] The step of obtaining a biological sample containing extracellular vesicles of the present invention may include ultracentrifugation, in which extracellular vesicles are precipitated by applying a centrifugal force of 100,000 × g or more, thereby removing contaminants smaller than extracellular vesicles and concentrating the extracellular vesicles.

[0035] 2. Electrical Conductivity Adjustment Step In this step, the electrical conductivity of the biological sample prepared in the previous step 1 is adjusted to 4 mS / cm or more and 15 mS / cm or less. The electrical conductivity is preferably adjusted by adjusting the salt concentration. Generally, biological samples have an osmotic pressure similar to that of physiological saline, which facilitates cell survival. The concentration and osmotic pressure of physiological saline are 0.154 mmol / L and 308 mOsm / L, respectively. The osmotic pressure of a biological sample is largely due to ionic components in the solution. Therefore, a positive correlation is observed between the electrical conductivity of the biological sample and the salt concentration, or between the electrical conductivity and osmotic pressure. By adjusting the electrical conductivity to 15 mS / cm or less, the adsorption efficiency of extracellular vesicles to positively charged carriers can be increased, since extracellular vesicles have a relatively weak negative charge. On the other hand, adjusting the electrical conductivity to 4 mS / cm or more can suppress the deterioration of extracellular vesicles. The electrical conductivity is preferably 9 mS / cm or more and 14 mS / cm or less. If the electrical conductivity is 15 mS / cm or less, the concentration of negatively charged ions such as chloride ions in the salt-containing buffer solution in the biological sample is reduced, thereby reducing the proportion of ion exchange groups in the carrier that are ionically bonded to negatively charged ions such as chloride ions, and increasing the number of ion exchange groups effective for adsorbing negatively charged extracellular vesicles. The increased density of effective ion exchange groups on the carrier surface generates a zeta potential on the carrier surface that can attract extracellular vesicles, which have a lower diffusion coefficient than low-molecular-weight ions, to the carrier surface. This makes it easier for extracellular vesicles, especially those with a low negative charge, to be adsorbed to the carrier. On the other hand, if the electrical conductivity is 4 mS / cm or more, the osmotic pressure of the aqueous solution components in the sample is not excessively reduced, thereby preventing damage to the extracellular vesicles due to the osmotic pressure difference between the inside and outside of the lipid bilayer membrane of the extracellular vesicles. Therefore, it is important to adjust the electrical conductivity to the appropriate range described above. When the electrical conductivity is 9 mS / cm or more and 14 mS / cm or less, the NaCl concentration in an aqueous NaCl solution (containing 10 mM Tris-HCl) is converted to 79 mmol / L or more and 130 mmol / L or less.

[0036] The step of reducing the electrical conductivity or salt concentration of a biological sample containing extracellular vesicles is not particularly limited, and can be carried out by diluting the biological sample with a buffer solution having a lower electrical conductivity or salt concentration than that of the biological sample. Examples of buffer solutions that can be used in this case include approximately 10 mM Tris-hydrochloride buffer (Tris-HCl), phosphate-buffered saline (PBS), and Tris-buffered saline (TBS). In another embodiment, the electrical conductivity or salt concentration of a biological sample containing extracellular vesicles can be adjusted by replacing the buffer solution by ultrafiltration or by desalting by dialysis.

[0037] 3. Contact Step with Carrier In this step, the biological sample whose electrical conductivity has been adjusted in the previous step 2 is brought into contact with a positively charged carrier. The carrier used in this step is not particularly limited, but cellulose-based resins such as cellulose acetate and cellulose triacetate, polysulfone-based resins, etc. are preferred. The carrier may also be a sheet-like separation membrane such as a flat membrane, a hollow fiber-like separation membrane (hollow fiber membrane), or a particulate porous or non-porous body.

[0038] In the present invention, the method for imparting a positive charge to the carrier is not particularly limited, but includes a method of imparting a cationic compound after forming the carrier. More specifically, a method of imparting a positive charge by contacting a carrier having hydroxyl groups with glycidyl trimethylammonium or glycidyl diethylamine is also available. Alternatively, a column packed with commercially available diethylaminoethyl (DEAE) cellulose resin may be used. DEAE cellulose is an anion exchanger in which diethylaminoethyl groups have been introduced into cellulose, and is available from Sigma-Aldrich, etc.

[0039] In the present invention, it is preferable to use, as the positively charged carrier, a cellulose-based ion exchange membrane in which at least a portion of the hydroxyl groups or acetyl groups at any of the 2-, 3-, and 6-positions of a porous substrate membrane containing a cellulose-based polymer are substituted with a compound having a positive charge.

[0040] Porous substrate membranes containing cellulose-based polymers can be produced by a wet method. In the wet method, a cellulose-based polymer is mixed with a solvent and a non-solvent to prepare a membrane-forming solution, which is then extruded into a hollow space and introduced into a coagulation bath to cause phase separation. Alternatively, a dry method, a thermally induced phase separation method, a stretching method, or the like may be used. On the other hand, a flat membrane can be produced by dissolving a cellulose-based polymer in a solvent, uniformly applying the solution to a substrate such as glass, immersing the substrate in a coagulation solution to coagulate it, and then washing and, if necessary, drying the substrate.

[0041] The solvent used to prepare the membrane-forming solution is preferably a polar solvent such as γ-butyrolactone, N-methylpyrrolidone, or dimethylacetamide. The solvents may be used alone or in combination. If necessary, a non-solvent such as water, glycerin, ethylene glycol, triethylene glycol, polyethylene glycol 200, or polyethylene glycol 400 may be added. The mixing ratio of the solvent to the non-solvent in the membrane-forming solution (solvent / non-solvent ratio) is preferably 90 / 10 to 10 / 90.

[0042] The coagulation liquid is preferably a mixture of a solvent and a non-solvent such as water. The solvent / non-solvent ratio in the coagulation liquid is preferably matched to the solvent / non-solvent ratio in the membrane-forming solution. By matching the solvent / non-solvent ratios of the membrane-forming solution and the coagulation liquid, fluctuations in the composition of the coagulation liquid can be suppressed even during continuous membrane production. In the case of flat membranes, the weight ratio of N-methylpyrrolidone to water in the mixed solution of N-methylpyrrolidone and water is preferably in the range of 30:70 to 50:50. By using such a coagulation bath, the pore size of the membrane surface that first comes into contact with the coagulation bath becomes larger than the pore size of the membrane surface that comes into contact with the substrate, resulting in the formation of an asymmetric structure in the thickness direction. When the concentration of N-methylpyrrolidone in the coagulation bath is 25% by mass or more but less than 30% by mass, the pore size of the membrane surface that first comes into contact with the coagulation bath becomes equal to the pore size of the membrane surface that comes into contact with the substrate, tending to alleviate the asymmetric structure of the membrane. Furthermore, if the concentration of N-methylpyrrolidone in the coagulation bath is less than 25% by mass, a dense layer without clear pores is likely to form on the membrane surface that first comes into contact with the coagulation bath. The temperature of the coagulation liquid is preferably 5 to 60°C.

[0043] The obtained porous substrate membrane is washed with warm water to remove excess solvent, etc. The washed porous substrate membrane may be stored in water, or may be dried after filling the pores with a pore-retaining agent such as an aqueous glycerin solution.

[0044] The obtained porous substrate membrane is then subjected to a treatment to introduce ion exchange groups. Specifically, after a deacetylation treatment, a treatment to impart a charge to the hydroxyl groups is performed. As an example, a method for producing a cationic cellulose membrane, which is an ion exchange membrane, by substituting positively charged groups for the hydroxyl groups of a cellulose-based porous substrate membrane is performed by immersing the cellulose-based porous substrate membrane in a non-dissolving solvent in the presence of alkali, dropping a solution of a positively charged compound, reacting under heating conditions, and quenching with alcohol.

[0045] The non-dissolving solvent is not particularly limited as long as it is the solvent that the cellulose-based porous substrate membrane does not dissolve and the solvent that the compound having positive charge dissolves.For example, water, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) etc. are good examples, and can be used alone or mixed according to the solubility of raw material.

[0046] The alkali used for deacetylation can be hydroxides such as lithium hydroxide, potassium hydroxide, sodium hydroxide, and cesium hydroxide, as well as carbonates and organic amines. Among these, sodium hydroxide is preferred because it is industrially inexpensive. The concentration of the alkali used is preferably in the range of 0.05% to 5.0% by mass. An alkali concentration of 0.05% by mass or higher can increase the amount of charged groups introduced, thereby more reliably increasing the amount of extracellular vesicles adsorbed per unit volume. On the other hand, an alkali concentration of 5.0% by mass or lower can suppress the degree of membrane swelling, thereby more reliably maintaining the structure (pore size) and physical properties (membrane strength) of the base membrane in the ion exchange membrane after the introduction of charged groups. Therefore, the alkali concentration is more preferably 0.1 to 2.5% by mass, and even more preferably 0.2 to 2.0% by mass.

[0047] The amount of charged groups imparted to the cellulose membrane can be controlled by the amount of the positively charged compound added. For example, when an epoxy group-containing tertiary amine compound is used, the concentration of the tertiary amine compound in the non-dissolving solvent is preferably in the range of 0.01 mol / L to 3.0 mol / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.9 meq / g per cellulose membrane weight. When an epoxy group-containing quaternary ammonium compound is used, the concentration of the quaternary amine compound in the non-dissolving solvent is preferably in the range of 0.01 mol / L to 1.0 mol / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.4 meq / g per cellulose membrane weight. If the amount of positively charged groups imparted is too high, the content of positively charged groups per polymer molecule will be high, resulting in increased water solubility and possibly weakening the membrane strength in water. Furthermore, if the amount of positively charged groups imparted is too low, the capture efficiency of extracellular vesicles may be reduced.

[0048] In the present invention, the positively charged carrier, such as an ion exchange membrane, is preferably in the form of a device housed in a container equipped with an inlet for introducing a liquid to be treated and an outlet for discharging the biological sample, which is the liquid to be treated after ion exchange treatment. This device has a first chamber and a second chamber separated by an ion exchange membrane, and the biological sample, which is the liquid to be treated, introduced into the first chamber permeates the ion exchange membrane and moves to the second chamber. During this process, extracellular vesicles in the biological sample, which is the liquid to be treated, are adsorbed to the surface and pore surfaces of the ion exchange membrane.

[0049] In the present invention, the ion exchange membrane, which is a carrier having a positive charge, may be a cellulose-based ion exchange membrane in which at least a portion of the hydroxyl groups or acetyl groups at any of the 2-, 3-, and 6-positions of the cellulose-based polymer are substituted with a compound having a positive charge.

[0050] In the present invention, examples of the cellulose-based polymer include cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate laurate, cellulose acetate oleate, and cellulose acetate stearate, among which cellulose acetate is preferred from the viewpoint of ease of introduction of a positively charged compound.Cellulose acetates with different degrees of acetylation and molecular weights are commercially available, and it is more preferred to use cellulose acetate and / or cellulose triacetate with a degree of acetylation of about 52 to 62.

[0051] In the present invention, as the compound having a positive charge, it is preferable to use a tertiary amine represented by the following formula (I) or a quaternary ammonium compound represented by the following formula (II): 1 ~R 3 are the same or different hydrogen atoms, or linear or branched alkyl groups having 1 to 10 carbon atoms, and n in the methylene group is an integer of 1 to 5. In the formula, X represents one or more selected from the group consisting of halogen atoms such as fluorine, chlorine, bromine, and iodine; leaving groups such as sulfonate esters such as tosylate, triflate, and mesylate; silyl groups such as alkoxysilyl and silanol; epoxide groups; isocyanate groups; and carboxylic acid groups. Any substituent X may be used for the compound having a positive charge, but from the perspective of ease of availability of raw materials, compounds in which X is chlorine, a silyl group, or an epoxide group are preferred.

[0052]

[0053]

[0054] In the present invention, the cellulose-based ion exchange membrane may be a so-called homogeneous membrane, in which the pore size is approximately constant from one surface to the other, or a so-called asymmetric membrane, in which the pore size changes continuously or discontinuously from one surface to the other. Furthermore, the minimum pore size of the ion exchange membrane is preferably 50 nm or more and 1000 nm or less. In an asymmetric membrane, the minimum pore size layer is preferably located near one of the surfaces. A large minimum pore size may reduce the probability of contact of extracellular vesicles such as exosomes with the membrane surface and / or pore surface, resulting in a low adsorption rate of extracellular vesicles in the treated liquid (biological sample). Furthermore, it may be impossible to remove cell debris. On the other hand, a small minimum pore size may result in a low recovery rate of extracellular vesicles that have penetrated the membrane, or may require a long recovery time. The minimum pore size of the ion exchange membrane is defined as the polystyrene particle size at which a rejection rate of 80% or more is achieved when measured using a polystyrene particle dispersion.

[0055] Furthermore, the average pore size of the surface of the ion exchange membrane, for example, the surface on the side not having the minimum pore size layer, is preferably 100 nm or more and 5000 nm or less. If the average pore size of the surface is 100 nm or more, biological samples can be processed at a sufficiently fast rate. Furthermore, if the average pore size of the surface is 5000 nm or less, the adsorption of extracellular vesicles to the carrier can be sufficiently promoted, making it possible to maintain the recovery rate. Furthermore, if the average pore size of the surface is within the above range, the effect of depth filtration can be more reliably exerted. The average pore size of the surface can be calculated using image processing software Image J based on photographs of both sides of the membrane taken with a scanning electron microscope (SEM) at a magnification of 1,000 to 20,000 times.

[0056] In this process, the biological sample to be treated may be treated by cross-flow or dead-end. Furthermore, when using an asymmetric membrane, the biological sample to be treated may be introduced into the large-pore side or the small-pore side. For example, for biological samples with a low solid content and a high extracellular vesicle concentration, such as culture supernatant, by using the large-pore side as the primary side and the small-pore side as the secondary side, clogging of the membrane pores by the adsorbed extracellular vesicles can be prevented even when the biological sample is loaded onto the membrane and the extracellular vesicles are charged and adsorbed to the membrane. On the other hand, for biological samples with a relatively high solid content relative to the extracellular vesicle concentration, such as serum, plasma, urine, and milk, by using the small-pore side as the primary side and the large-pore side as the secondary side, impurities can be removed by surface filtration and membrane clogging due to complete pore blockage can be prevented.

[0057] In the present invention, the ion exchange capacity of the positively charged carrier is preferably 0.05 meq / g or more and 1.5 meq / g or less. If the ion exchange capacity is 0.05 meq / g or more, the zeta potential of the membrane is stable, and even substances to be purified with a small diffusion coefficient, such as extracellular vesicles, can be sufficiently attracted to the membrane, and the adsorption amount is sufficiently large, so a large-capacity device is not required. On the other hand, if the ion exchange capacity is 1.5 meq / g or less, the membrane strength is sufficient, and the adsorption amount per unit volume of the membrane is appropriate, thereby preventing membrane clogging. When a tertiary amine compound is used, the ion exchange capacity is preferably 0.03 meq / g or more and 0.9 meq / g or less per carrier mass. When a quaternary ammonium compound is used, the ion exchange capacity is preferably 0.03 meq / g or more and 0.4 meq / g or less per carrier mass.

[0058] In the present invention, the carrier may be in the form of a flat membrane or a hollow fiber membrane, but the thickness of the membrane is preferably 10 μm to 1000 μm. If the thickness of the membrane is 10 μm or more, the strength of the membrane is sufficiently high, and handling during membrane production and device fabrication is highly convenient. On the other hand, if the thickness of the membrane is 1000 μm or less, the recovery time of extracellular vesicles can be reduced and the recovery rate can be maintained. Furthermore, in the case of a hollow fiber membrane, the inner diameter is preferably 50 μm to 1000 μm. If the inner diameter is 50 μm or more, the shear stress of the liquid flowing through the hollow portion is sufficiently small, and damage to extracellular vesicles can be sufficiently suppressed. On the other hand, if the inner diameter is 1000 μm or less, the membrane area per device can be sufficiently large.

[0059] In the present invention, the zeta potential of the carrier is preferably in the range of 10 mV to 70 mV under conditions of pH 6.5 to 7.5. If the zeta potential is 10 mV or higher, the adsorptive power for extracellular vesicles with a low diffusion coefficient is sufficiently high, ensuring adsorption. If the zeta potential is 70 mV or lower, the membrane strength is sufficiently high, and when particles such as extracellular vesicles are to be purified, the amount of adsorption per unit membrane volume becomes appropriate, preventing membrane clogging. The zeta potential of a sheet-like carrier can be measured using a flat-plate zeta potential measurement cell.

[0060] In the present invention, the specific surface area of ​​the support is such that the total specific surface area of ​​pores having a pore diameter of 30 nm or more is 3 m 2 / g or more is preferable, and 8m 2 / g or more is more preferable. 2 / g or more, a sufficient amount of extracellular vesicles can be adsorbed. Specific surface area is generally measured by gas adsorption or mercury porosimetry. Gas adsorption can detect the specific surface area of ​​micropores of approximately 2 nm or less and mesopores of 2 to 50 nm, whereas mercury porosimetry can detect mesopores and macropores of 50 nm or more. The larger the specific surface area, the better in order to ensure the amount of extracellular vesicles adsorbed. However, since extracellular vesicles cannot enter pores with a pore diameter of less than 30 nm, the specific surface area consisting of mesopores and macropores with a pore diameter of 30 nm or more must be sufficiently large in order to increase the amount of extracellular vesicles adsorbed per unit volume of membrane. While a specific surface area of ​​pores that is too large does not cause problems, a specific surface area of ​​150 m 2 / g or less is preferable, and 120m 2 / g or less is more preferable.

[0061] The porosity of the carrier is preferably 40% or more and 90% or less. If the porosity is 40% or more, a sufficient area for adsorption of extracellular vesicles can be secured. If the porosity is 90% or less, the strength of the carrier can be secured sufficiently. The porosity is more preferably 45% or more and 90% or less.

[0062] When using the carrier of the present invention for the separation and purification of extracellular vesicles, it is preferable to adjust the zeta potential, specific surface area, porosity, etc. within specific ranges. Among extracellular vesicles, exosomes are extracellular vesicles with a size of 30 nm to 150 nm and a negative charge on the surface. For example, when efficiently adsorbing and desorbing exosomes from biological samples such as cell culture supernatants, if the pores are too small and the number of micropores is too large, exosomes cannot enter the pores, and increasing the specific surface area of ​​the carrier will not increase the adsorption area or adsorption capacity. Furthermore, if the pores are too large and the number of macropores is too large, the distance between the membrane surface and exosomes will be too great, making it difficult for electrical attraction to occur, or extracellular vesicles with a diameter exceeding 150 nm will enter the pores, reducing the adsorption efficiency of the target exosomes. On the other hand, it may be preferable for ion exchange membranes to have a certain degree of micropores. Biological samples and cell culture supernatants contain many small molecular weight substances, many of which are electrically charged. By adsorbing such small molecular weight substances into micropores, it becomes possible to effectively utilize the adsorption sites of mesopores and macropores for adsorption of exosomes. The specific surface area of ​​pores with a pore diameter of less than 30 nm is 30 m 2 / g or more 80m 2 / g or less is preferred.

[0063] The ion exchange capacity of the positively charged carrier is preferably 0.08 meq / g or more and 0.3 meq / g or less. If the ion exchange capacity is 0.3 meq / g or less, the content of positively charged groups per polymer molecule constituting the carrier is not too high, and the water solubility of the carrier is not excessively high, so that the carrier strength in water can be sufficiently ensured. Furthermore, if the ion exchange capacity is 0.08 meq / g or more, the capture efficiency of extracellular vesicles can be more reliably maintained at a high level. The positively charged group is preferably a tertiary amine group or a quaternary ammonium group. Diethylamine can be used as the tertiary amine, and trimethylammonium can be used as the quaternary ammonium.

[0064] In the present invention, "contacting" refers to immersing the carrier in the biological sample. Furthermore, when the carrier has a porous membrane structure, it also includes creating a pressure difference between the primary side and secondary side of the membrane so that the biological sample penetrates into the porous membrane, thereby passing the biological sample through the interior of the porous membrane from the primary side to the secondary side of the membrane. Furthermore, when the carrier is a particulate resin, it also includes passing the biological sample from the inlet to the outlet of a column packed with the resin by a pressure difference. The amount of extracellular vesicle particles that can be processed by the carrier depends on the amount of residual medium components in the culture supernatant, but when the anion exchange capacity of the carrier is 0.08 to 0.3 meq / g, it is approximately 10 9 ~10 12 The amount of the biological sample to be treated must be adjusted so that it does not exceed this amount relative to the amount of the carrier.

[0065] 4. Washing Step In the present invention, it is preferable to bring a biological sample into contact with a positively charged carrier to adsorb extracellular vesicles and the like onto the carrier, and then wash the carrier by contacting it with a buffer solution. The buffer solution is not particularly limited, but Tris-HCl is preferred. Other buffer solutions include TBS and PBS. The salt concentration of the buffer solution is preferably adjusted to be equal to or lower than the salt concentration of the biological sample to prevent the target extracellular vesicles from detaching from the carrier and eluting together with other impurities. Specifically, it is preferably 50 mmol / L to 150 mmol / L and 6.08 mS / cm to 16.01 mS / cm. The pH is not particularly limited, but is preferably in the range of 7 to 8. The volume of the buffer solution used for washing is preferably 5 to 100 times the volume of the column or porous body.

[0066] 5. Recovery Step In this step, the carrier onto which extracellular vesicles have been adsorbed in the previous steps 3 and 4 is contacted with a buffer solution containing a metal salt to dissociate and recover the extracellular vesicles from the carrier. In the present invention, to elute and recover extracellular vesicles such as exosomes adsorbed and bound to the carrier, contact is performed with a buffer solution containing a monovalent or divalent metal salt. The salt concentration of the buffer solution is preferably 300 mmol / L to 800 mmol / L and 30.91 mS / cm to 80.58 mS / cm. Depending on the purpose, such as recovering extracellular vesicles by type based on differences in the charge of the extracellular vesicles, contact with the carrier while increasing the salt concentration, such as by stepwise elution or linear gradient elution, may be preferable. The monovalent or divalent metal salt is not particularly limited, but NaCl is preferred. In other embodiments, the metal salt may be MgCl, NaSO, or MgSO. As the buffer solution, Tris-HCl, PBS and TBS are preferred, and Tris-HCl is more preferred.

[0067] In the present invention, the salt concentration and electrical conductivity of a sample containing extracellular vesicles may be adjusted to enhance the storage stability of the recovered extracellular vesicles. The salt concentration and electrical conductivity may be in a range close to the electrolyte concentrations in a living body, preferably 140 mmol / L or more and 160 mmol / L or less, and more than 15 mS / cm and 17.01 mS / cm or less. The electrical conductivity is more preferably 15.01 mS / cm or more. An example of a method for measuring the salt concentration is to measure the electrical conductivity of the recovered fraction and calculate the NaCl concentration (Figure 2). Based on the obtained salt concentration, dilution may be performed by adding 10 mmol / L Tris-HCl (pH 7.5). Other methods include buffer replacement by ultrafiltration or desalting by dialysis.

[0068] In the present invention, "concentration" refers to reducing the volume of a solution containing the target extracellular vesicles and increasing the particle concentration of the target extracellular vesicles.

[0069] This application claims the benefit of priority based on Japanese Patent Application No. 2023-149357, filed on September 14, 2023. The entire contents of the specification of Japanese Patent Application No. 2023-149357, filed on September 14, 2023, are incorporated herein by reference.

[0070] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples. The physical properties and other properties in the following examples were evaluated using the following methods.

[0071] Test Example 1: Measurement of ion exchange capacity of carrier The ion exchange capacity of the carrier was measured as follows. A 0.1% by mass aqueous solution of bromophenol blue (BPB) was prepared, and a positively charged carrier was immersed in the solution at room temperature for 1 hour to stain it. After the dyeing, the carrier was washed with water and then immersed in an 8% by mass aqueous sodium chloride solution to elute the BPB adsorbed on the carrier. The absorbance at 590 nm of the sodium chloride aqueous solution was measured, and the number of moles of BPB adsorbed per mass of the carrier was calculated based on a calibration curve for a BPB aqueous solution containing 8% by mass sodium chloride, and this was used as the ion exchange capacity.

[0072] Test Example 2: Evaluation of Particle Rejection The rejection of a porous substrate membrane and an ion exchange membrane was evaluated using a dispersion of polystyrene particles. A Tween 20 aqueous solution containing 0.01% by mass of polystyrene particles of various sizes dispersed therein was passed through the membrane at an operating pressure of 10 kPa to obtain a permeate. The polystyrene particle concentrations in the original solution and the permeate were measured by absorbance at 250 nm, and the rejection was calculated using the following formula: Rejection (%) = {1 - (Ca - Cb) / Ca} x 100, where Ca is the polystyrene particle concentration in the original solution and Cb is the polystyrene particle concentration in the permeate. Rejection was measured using polystyrene particles with particle sizes of 79, 132, 208, 262, 313, 420, 460, and 616 nm, and the polystyrene particle size at which the rejection rate reached 80% was defined as the minimum pore size.

[0073] Test Example 3: Observation of film structure The film to be evaluated was placed on a sample stage for SEM, dried at room temperature, platinum was vapor-deposited, and observed under a scanning electron microscope (SU-1500, manufactured by Hitachi High-Technologies Corporation).

[0074] Test Example 4: Measurement of the average pore diameter on the large pore side of the membrane The average pore diameter on the large pore side of the surface SEM image of the membrane observed at 10,000x magnification was measured by the following procedure. Open the image on the image processing software Image J, draw a line within the range of the scale bar using the straight line tool from the toolbar, select Analyze> Set Scale, enter the length of the scale bar in "Known Distance" and the unit in "Unit of length". In Image> Adjust> Auto Threshold, set the Method mode to Default, select OK and perform binarization. In Analyze> Set Measurements, confirm that "Feret's diameter" is checked and press OK. In Analyze > Analyze Particles, set "Size (pixel^2)" to 0-Infinity, confirm that the "Display results," "Exclude on edges," and "Include holes" boxes are checked, select OK, and obtain Feret diameter measurement data on the Results screen. To eliminate noise below 15 pixels, the length equivalent to 15 pixels was calculated in the same units as the Feret diameter, and values ​​below that were eliminated to calculate the average Feret diameter.

[0075] Test Example 5: Measurement of zeta potential The zeta potential of the membrane was calculated by attaching a flat plate zeta potential measurement cell ("ZEN1020" manufactured by Malvern Panalytical) to a Zetasizer Nano ZS and measuring the mobility of aluminum oxide particles in ion-exchanged water of pH 6.5 to 7.5.

[0076] Test Example 6: Measurement of Specific Surface Area Measurement was performed using a pore distribution analyzer ("Autopore IV9520" manufactured by Shimadzu Corporation) under the following conditions. The sample was cut into strips of approximately 12.5 mm x 25 mm, and 0.04 to 0.15 g was placed in a 5 mL powder cell (stem volume 0.4 mL). Measurement was performed under an initial pressure of approximately 3.7 kPa (corresponding to a pore diameter of approximately 340 μm). Mercury parameters were set to the instrument's default mercury contact angle of 130 degrees and mercury surface tension of 485 dynes / cm. The pore distribution was obtained by plotting the change in the log differential volume of mercury penetrating the sample against the pore diameter to obtain a pore distribution curve. The specific surface area was measured by measuring the pore volume V for each mode diameter D, and the specific surface area S for each mode diameter D was calculated using the following formula. The total specific surface area was calculated from the sum of the values ​​in the range of 0.03 to 500 μm. S=4V / D

[0077] Test Example 7: Nanoparticle Tracking Analysis The average particle number and diameter of the recovered extracellular vesicles were measured by nanoparticle tracking analysis (NTA) using a nanoparticle analysis system ("NanoSight NS300" manufactured by Malvern Panalytical). The camera level was set to 14 and the detection threshold was set to 7. When the salt concentration of the culture supernatant was changed, the particle number was measured at least 24 hours after the change.

[0078] Test Example 8: Western Blotting Western blotting was performed to detect extracellular vesicle proteins, including surface proteins of the recovered extracellular vesicles. The sample containing the recovered extracellular vesicles was concentrated as necessary, dissolved in a sample buffer ("NuPage LDS Sample Buffer" manufactured by Thermo Fisher Scientific), and boiled at 95°C for 5 minutes. Note that a sample buffer further containing 100 mmol / L dithiothreitol was used for detecting TSG101, β-actin, and GAPDH, and a sample buffer not containing dithiothreitol was used for detecting CD63 and CD81. Each sample and molecular weight marker ("MagicMark XP Western Protein Standard" manufactured by Thermo Fisher Scientific) were separated by 10% polyacrylamide gel electrophoresis using SDS running buffer (Tris / Glycine / SDS Buffer) at 30 mA (one gel) or 40 mA (two gels) for 60 minutes. The resulting gel was transferred to a polyvinylidene fluoride membrane using blotting buffer ("EzFastBlot" manufactured by ATTO). After blocking with a blocking agent ("Blocking One" manufactured by Nacalai Tesque), each membrane was reacted with a primary antibody for 1 hour. After washing, the membrane was reacted with a secondary antibody for 1 hour. After washing, bands derived from the target protein were visualized using an HRP detection reagent ("EzWestLumi plus" manufactured by ATTO).

[0079] Example 1 (1) Biological Sample Acquisition Process A human fetal kidney-derived cell line (HEK293.2sus) was used as the cultured cells. HEK293.2sus was obtained from American Type Culture Collection, Inc. HEK293.2sus cells were cultured in 293 SFM II medium (ThermoFisher Scientific) in a 100 mL Erlenmeyer flask (Corning) at 100 rpm, 8% CO2 concentration, and 5 x 10 5 The cells were cultured at 70 cm 2The cells were cultured in 293 SFM II medium in a 5% CO2 culture flask (Corning). The culture medium was changed every 3 or 4 days, and the culture was continued for a final 17 days. The culture medium obtained when the medium was changed was collected, pooled, and used for exosome preparation. The collected culture medium was centrifuged at 4°C at 300 x g for 10 minutes, followed by 10,000 x g for 20 minutes, and the supernatant was collected. The culture medium was then filtered through a 0.45 μm filter (Corning) to prepare the culture supernatant.

[0080] (2) Preparation of anion exchange membrane: A cellulose acetate membrane filter (Advantec) with a nominal pore size of 0.2 μm was used as a positively charged carrier. It was deacetylated by immersing it in a 0.1 M aqueous solution of sodium hydroxide at room temperature for 4 hours. The resulting deacetylated membrane was then immersed in a 0.4 M aqueous solution of glycidyl diethylamine at 65° C. for 4 hours to obtain an ion exchange membrane. The performance of the resulting ion exchange membrane is shown in Table 1.

[0081]

[0082] (3) Equilibration Step 5 mL of 10 mmol / L Tris-HCl (pH 7.5) (equilibration buffer) containing 100 mmol / L (11.05 mS / cm) NaCl was passed through an anion exchange membrane having a diameter of 25 mm to equilibrate the anion exchange membrane.

[0083] (4) Permeation Step NaCl was dissolved in 10 mmol / L Tris-HCl buffer, and a calibration curve showing the relationship between NaCl concentration and electrical conductivity was created. Electrical conductivity was measured using an electrical conductivity meter ("LAQUAtwin EC-33" manufactured by HORIBA). The measurement section was washed twice with the measurement sample before measurement. 3 mL of 10 mmol / L Tris-HCl buffer was added to 6 mL of culture supernatant filtered through a 0.45 μm membrane filter, and the culture supernatant with an electrical conductivity of 11.05 mS / cm was passed through the membrane.

[0084] (5) Elution step After passing 10 mL of 10 mmol / L Tris-HCl buffer through the membrane to wash the membrane, 2 mL of 300 mmol / L (30.91 mS / cm) NaCl was passed through the membrane to elute the extracellular vesicles bound to the membrane, thereby obtaining a purified fraction of extracellular vesicles.

[0085] (6) Isotonicity Step Next, the electrical conductivity of the fraction from which extracellular vesicles were eluted was measured. The NaCl concentration was calculated from the electrical conductivity value using the calibration curve (Figure 2), and then 10 mmol / L Tris-HCl (pH 7.5) was immediately added to the solution to make it isotonic with 10 mmol / L Tris-HCl (pH 7.5) containing 0.15 mol / L NaCl, yielding an extracellular vesicle solution. Note that the equilibration to isotonicity steps were all performed with the solution cooled to 4 to 10°C.

[0086] (7) Characterization The samples containing the fractionated extracellular vesicles were subjected to NTA analysis to measure the average particle number and diameter, and the presence of extracellular vesicles was confirmed by Western blotting to detect CD63. NTA analysis was performed using a nanoparticle analysis system ("NanoSight NS300" manufactured by Malvern Panalytical) at a camera level of 14 and a detection threshold (DT) of 7. Western blotting was performed using an anti-CD63 mouse antibody (MEX002-3, MBL) as the primary antibody and an Anti-Mouse IgG HRP-Linked Whole Ab Sheep (Cytiva) as the secondary antibody, with iBright TM Detection was performed using an FL1500 Imaging System (ThermoFisher scientific).

[0087] Comparative Example 1 An extracellular vesicle solution was obtained in the same manner as in Example 1, except that in the permeation step, the electrical conductivity of the culture supernatant was not adjusted and the culture supernatant with the same salt concentration was permeated through the membrane. In this case, the electrical conductivity of the culture supernatant that permeated the anion exchange membrane was 16.01 mS / cm.

[0088] Comparative Example 2 An extracellular vesicle solution was obtained in the same manner as in Example 1, except that in the permeation step, an equilibration buffer was added to the culture supernatant to prepare a culture supernatant with an electrical conductivity of 3.10 mS / cm, and the culture supernatant was permeated through the membrane.

[0089]

[0090] The results in Table 2 show that in Comparative Example 1, in which the culture supernatant was purified without adjusting its electrical conductivity to 16.01 mS / cm, the number of particles recovered from the carrier with ion exchange groups was reduced. This indicates that the high salt concentration of the culture supernatant prevented sufficient adsorption of extracellular vesicles to the carrier, resulting in their leakage into the permeate (flow-through) fraction. Furthermore, in Comparative Example 2, in which the electrical conductivity of the culture supernatant was set to 3.10 mS / cm, the results of particle measurement using NTA in the culture supernatant significantly increased. This indicates that the extracellular vesicles had ruptured and been damaged. In Comparative Example 2, significant alteration of the extracellular vesicles was confirmed, so a permeation test was not performed. Furthermore, the results of a Western blotting test are shown in Figure 3. CD63 is known as a marker for EVs, and it has been reported that it is particularly expressed in exosomes among EVs (Borges FT, et al., 2013, Extracellular vesicles: structure, function, and potential clinical uses in renal diseases, Braz J Med Biol Res, 46(10), 824-30). It has also been reported that exosome-rich fractions have a weaker negative charge than other EVs such as MVs (Seo N, et al., 2022, Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method, J Extracell Vesicles, Mar, 11(3)). As shown in Figure 3, the percentage of CD63-positive particles per unit particle in the purified fraction was higher in Example 1 than in Comparative Example 1. This suggests that by using the method disclosed in this specification, it is possible to adsorb a larger amount of exosomes, which have a particularly weak negative charge, from among the particles in the culture supernatant to the membrane, thereby reducing loss and enabling recovery.

[0091] According to the present invention, extracellular vesicles contained in a biological sample can be easily purified and recovered with a high recovery rate. The extracellular vesicles obtained by the present invention can be suitably used in fields such as drug delivery, pharmaceuticals, in vitro diagnostics, food, and cosmetics.

[0092] 1, 2: Liquid medium storage container 3: Cell collection container 4, 5, 6, 7, 8, 9, 10, 11, 12: Valve 13, 14, 15, 16: Sterile connection connector 17: Cell culture container 18: Waste liquid collection container 19, 20: Liquid transfer pump 21: Operation controller (operation panel) 31: CO2 incubator

Claims

1. A method for producing extracellular vesicles, comprising the steps of: (a) obtaining a biological sample containing extracellular vesicles; (b) adjusting the electrical conductivity of the biological sample to between 4 mS / cm and 15 mS / cm; (c) contacting the biological sample with a positively charged carrier; and (d) contacting the carrier with a buffer solution containing a metal salt to recover extracellular vesicles.

2. The method of claim 1, wherein step (a) comprises isolating cells from an animal or thawing frozen cells of a cell stock, followed by culturing.

3. The method according to claim 1, further comprising a step of removing at least a portion of contaminants from the biological sample between steps (a) and (b).

4. The method according to claim 3, wherein the step of removing at least a portion of the impurities is one or more selected from the group consisting of centrifugation, microfiltration, ultrafiltration and dialysis.

5. The method according to claim 1, wherein in step (c), the carrier is a separation membrane having anion exchange capacity.

6. The method according to claim 5, characterized in that in step (c), the support has a tertiary amine and / or a quaternary ammonium group.

7. The method according to claim 1, further comprising a step of contacting the carrier with a buffer solution and washing the carrier between steps (c) and (d).

8. The method according to claim 1, wherein in step (d), the buffer solution containing the metal salt is any one of Tris-HCl buffer, phosphate buffered saline, and Tris buffered saline.

9. The method of claim 1, wherein in step (d), the metal salt is NaCl.

10. The method according to claim 1, characterized in that in step (d), the method includes a step of adjusting the electrical conductivity of the sample containing the recovered extracellular vesicles to greater than 15 mS / cm.

11. The method according to claim 1, wherein the extracellular vesicles are exosomes and / or microvesicles.

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