Methods for separating and purifying extracellular vesicles

Abstract

To provide a separating and purifying method for separating and purifying extracellular vesicles.SOLUTION: A method for separating and purifying extracellular vesicles from solution including the extracellular vesicles and unnecessary matters. The method for separating and purifying is an adsorbent treatment method in which adsorbent is allowed to adsorb and hold unnecessary matters from which the extracellular vesicles are removed by bringing solution including the extracellular vesicles and unnecessary matters in contact with the adsorbent, where the adsorbent is porous particulate in which the inside of a pore is positively charged, and can adsorb negatively charged unnecessary matters from which the extracellular vesicles are removed and held inside the pore.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a separation and purification method for separating and purifying extracellular vesicles.

Background Art

[0002] As a method for separating and purifying useful substances from a culture solution, a method using a separation membrane is known. Patent Document 1 describes an invention of a method for concentrating a unicellular algal culture solution by cross-flow filtration using a hollow fiber ultrafiltration membrane (UF membrane) module with a fractional molecular weight of 10,000 to 1,000,000, and performing periodic countercurrent washing. When the culture solution is concentrated by cross-flow filtration, a concentrated solution exists outside the UF membrane, and a permeate enters the inside of the UF membrane. When performing countercurrent washing, the washing water enters the inside of the UF membrane and then exits the outside of the UF membrane, thereby washing the UF membrane.

[0003] Patent Document 2 includes a bleeding step of discharging a culture solution from a cell culture tank and adding the same amount of fresh medium as the discharged culture solution to the culture tank, and a filtration step of filtering the culture solution extracted from the culture tank using a porous membrane having substantially no dense layer. The filtration in the filtration step is tangential flow filtration, and the velocity of the permeate in the filtration step is 1.0 LMH or less. An invention of a method for recovering useful substances is described. It is described that the useful substance is selected from the group consisting of proteins, viruses, exosomes, and nucleic acids. It is described that the tangential flow filtration can also be alternating tangential flow filtration. Extracellular vesicles are a general term for particles without a nucleus covered by a lipid bilayer released outside the cell, and include nucleic acids, proteins, lipids, various metabolites, etc., and exosomes, microvesicles, apoptotic vesicles, etc. correspond to them.

[0004] Non-patent document 1 describes the ExoScreen method as a new method for detecting exosomes. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 3-39084 [Patent Document 2] Japanese Patent Publication No. 2018-76291 [Non-patent literature]

[0006] [Non-Patent Document 1] Cytometry Research 26(1):1~6, 2016, "A New Development in Liquid Biopsy Using Exosomes", Yusuke Yoshioka, Takahiro Ochitani [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of this disclosure is to provide a method for separating and purifying extracellular vesicles. [Means for solving the problem]

[0008] This disclosure relates to a method for separating and purifying extracellular vesicles from a solution containing extracellular vesicles and waste products, The separation and purification method is an adsorbent treatment method in which the culture supernatant of a solution containing the extracellular vesicles and unwanted materials is brought into contact with an adsorbent, thereby adsorbing and retaining the unwanted materials, excluding the extracellular vesicles, onto the adsorbent. The present invention provides a method for separating and purifying extracellular vesicles, wherein the adsorbent is a porous granular material with positively charged pores, and can adsorb and retain negatively charged unwanted substances, excluding the extracellular vesicles, within the pores.

[0009] Furthermore, this disclosure relates to a method for separating and purifying extracellular vesicles from a solution containing extracellular vesicles and waste products, The present invention provides a method for separating and purifying extracellular vesicles, wherein the separation and purification method is a combination of the above-described adsorbent treatment method and membrane separation method. [Effects of the Invention]

[0010] According to the method for separating and purifying extracellular vesicles described herein, extracellular vesicles can be concentrated to a high concentration. [Brief explanation of the drawing]

[0011] [Figure 1] A diagram showing a separation and purification apparatus used for separating and purifying extracellular vesicles. [Figure 2] A partially enlarged view of a separation and purification apparatus of a different embodiment than that shown in Figure 1, used in the separation and purification flow shown in Figure 1. [Modes for carrying out the invention]

[0012] One embodiment of a method for separating and purifying extracellular vesicles from a solution containing the extracellular vesicles and waste products of the present disclosure is described. The separation and purification method of the above embodiment is an adsorbent treatment method in which an adsorbent is brought into contact with a solution containing the extracellular vesicles and unwanted materials, thereby adsorbing and retaining the unwanted materials, excluding the extracellular vesicles, on the adsorbent. The solution containing the extracellular vesicles and waste products is preferably a culture supernatant containing extracellular vesicles, and more preferably a culture supernatant of mesenchymal stem cells containing extracellular vesicles. Mesenchymal stem cells containing extracellular vesicles can be derived from various sources, such as bone marrow, blood, fat, umbilical cord, umbilical cord blood, periosteum, perichondrium, and other somatic tissues. Methods for culturing them are described, for example, in Japanese Patent Publication No. 2011-67175 and Japanese Patent Publication No. 2003-52360.

[0013] The adsorbent is a porous granular material that can adsorb and retain impurities other than extracellular vesicles in a solution containing the extracellular vesicles and impurities, and examples include porous granular materials of known adsorbents, carriers for chromatography, filter paper, membranes, filters, hollow fibers, fibers, polymers that can be processed into nanofibers, and inorganic materials. The porous granular material has a large number of pores, the interior of the pores is positively charged, and it can adsorb and retain negatively charged impurities other than the extracellular vesicles inside the pores. The porous granular material is based on polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, and glucomannan, synthetic polymers such as polyacrylic acid or its derivatives, polyvinyl alcohol, nylon, polysulfone, polyacrylonitrile, polyethylene, polypropylene, and polystyrene, and materials based on glass, porous glass, silica gel, hydroxyapatite, etc. The porous granular material can be selected and used from those with an average pore diameter in the range of 0.1 to 1000 nm according to the type of the impurities, etc. The average pore diameter can be measured by mercury intrusion porosimetry or inverse size exclusion chromatography. The porous granular material can be selected and used from those with an average particle diameter in the range of 1 to 1000 μm according to the type of the impurities, etc. The average particle diameter can be measured by laser diffraction scattering method.

[0014] The method of contacting the culture supernatant of the solution containing the extracellular vesicles and impurities with the adsorbent is The method of passing the culture supernatant through a column filled with the adsorbent, and a method selected from the method of adding the adsorbent into a container containing the culture supernatant and then stirring and mixing, or a method obtained by modifying a part of each of the above methods can be used. For the contact method using the column, a plurality of columns can be used, and the plurality of columns can be used in combination with those having different inner diameters and lengths (that is, those having different filling amounts of the adsorbent). The contact method using the aforementioned containers can use multiple containers, and these multiple containers can be combined to have different internal volumes (i.e., different amounts of adsorbent added).

[0015] The method for separating and purifying extracellular vesicles from a solution containing extracellular vesicles and unwanted materials according to this disclosure can also be performed by using multiple adsorbents having different adsorption properties, and carrying out multiple adsorbent treatments using the multiple adsorbents. Multiple adsorbents having different adsorption properties can be used in combination of one or more selected from those with different pore sizes of porous granules, different specific surface areas of porous granules, different average particle diameters of porous granules, different types of substrates constituting the porous granules, and different types and densities of functional groups present on the inner surface of the pores of porous granules. For example, even if the specific surface area of ​​the porous granular material is the same, the type and density of functional groups present on the inner surface of the pores of the porous granular material will result in different positive charge states inside the pores, thus affecting the types of negatively charged unwanted substances that are easily adsorbed. When performing the adsorbent treatment method multiple times, the type and amount of adsorbent can be changed each time. It is preferable to use a combination of multiple adsorbents having different adsorption properties and to perform the procedure multiple times, as this enhances the ability to respond to changes in the type and amount of unwanted materials (adsorption retention capacity) in a solution containing extracellular vesicles and unwanted materials, excluding the extracellular vesicles. After performing the adsorbent treatment method, the adsorbent that has adsorbed and retained unwanted materials can be separated by methods such as filtration to obtain a treated liquid that has undergone the desired adsorbent treatment method.

[0016] Another embodiment of a method for separating and purifying extracellular vesicles from a solution containing the extracellular vesicles and waste products of this disclosure is described. The method for separating and purifying extracellular vesicles in the aforementioned alternative embodiment is a method that combines the adsorbent treatment method and membrane separation method described above. The method for separating and purifying extracellular vesicles in the aforementioned alternative embodiment may be a method in which the adsorbent treatment method and the membrane separation method are performed alternately, or a method in which they are performed randomly. However, in either case, it is preferable that the first treatment is the adsorbent treatment method. There are no particular restrictions on the material, shape, and pore size of the membrane used in the membrane separation method, and the membrane separation method can include total filtration and tangential flow filtration. For example, the membrane morphology can include flat membranes, hollow fiber membranes, and tubular membranes. The membrane separation method is preferably a membrane separation method in which a flat membrane microfiltration membrane is set in a syringe cylinder and the entire volume is filtered, or a membrane separation method that uses an ultrafiltration membrane in the form of a hollow fiber membrane for filtration. The hollow fiber ultrafiltration membrane used in the aforementioned membrane separation method preferably has an inner diameter of 0.2 mm to 1.4 mm and a molecular weight cutoff of 100,000 to 1,000,000. The filtration method in the hollow fiber type ultrafiltration membrane is preferably tangential flow filtration in one direction or alternating directions.

[0017] Next, we will explain a separation and purification method using the separation and purification apparatus 1 shown in Figure 1, in which a treated liquid is first obtained by an adsorbent treatment method, and then the treated liquid is treated by a membrane separation method. In the first step, the treated liquid, which has been treated by an adsorbent treatment method using an adsorbent, is placed into the first tank 10.

[0018] Although the first tank 10 is cylindrical in shape in Figure 1, it is not limited to this, and its shape and volume can be determined according to the conditions of the installation site and the processing volume. The first tank 10 is preferably transparent so that the liquid level inside can be observed visually, and is also preferably made of a water-repellent material to prevent liquid from adhering to and remaining on the inner wall surface of the first tank 10. The first tank 10 is preferably made of part or all of an acrylic resin such as polyacrylonitrile or polyacrylic acid ester, polycarbonate, or fluororesin. The first tank 10 may be provided with a dispensing line for extracting the final concentrated liquid from within the first tank 10.

[0019] As shown in Figure 2, the connection portion 11 between the first tank 10 and the hollow fiber membrane 30, including the liquid inlet / outlet 10a, has a conical inclined surface 11a and a cylindrical vertical surface 11b, with the diameter decreasing from the first tank 10 side towards the hollow fiber membrane 30 side. In Figure 2, the connecting portion 11 has a conical inclined surface 11a and a cylindrical vertical surface 11b, but if it has a conical inclined surface 11a, the cylindrical vertical surface 11b is not necessary. Having the connecting portion 11 shown in Figure 2 is preferable because it prevents the treated solution or its concentrate, treated by the adsorbent treatment method, from accumulating at the bottom of the first tank 10, thereby increasing the recovery rate of extracellular vesicles.

[0020] Prior to the first step, a pretreatment step using a microfiltration membrane (microfiltration membrane module) can be performed as needed, but this can be omitted since an adsorbent treatment method is already being used. The microfiltration membrane (microfiltration membrane module) is preferably one with a pore size of 0.1 μm to 0.5 μm. In the pretreatment process, the treated liquid, which has been treated by an adsorbent treatment method using an adsorbent, is filtered by a microfiltration membrane (microfiltration membrane module), and the resulting filtrate (pretreatment liquid) is sent to the first tank 10.

[0021] In the second step, with the on / off valve (such as an electromagnetic valve) 46 open, the buffer solution in the buffer solution tank 40 is supplied to the first tank 10 from the buffer solution supply line 45 to dilute the treated solution that has been treated by the adsorbent treatment method using an adsorbent. When the first tank 10 contains a diluted solution of the treated liquid processed by the adsorbent treatment method using an adsorbent, there is a space remaining at the top of the first tank 10 where the diluted solution is not present. The buffer in the buffer tank 40 is preferably a medical or biochemical buffer, and can be a phosphate buffer (PBS), Tris-HCl buffer, sodium citrate buffer, citrate-phosphate buffer, acetate buffer, borate buffer, etc. When replenishing the buffer solution in the buffer solution tank 40, the solution is replenished through the buffer solution replenishment line 41. Alternatively, the order of the first and second steps can be reversed: the buffer solution in the buffer solution tank 40 can be supplied to the first tank 10 via the buffer solution supply line 45, and then the treated liquid, which has been treated by the adsorbent treatment method using an adsorbent, can be added to the first tank 10. Alternatively, the first and second steps can be combined into a single step. A diluted solution can be prepared by adding and mixing the treated liquid, which has been treated by an adsorbent treatment method using an adsorbent, with a buffer solution in a separately provided mixing tank, and then adding the diluted solution to the first tank 10.

[0022] In the third step, a pump (not shown) or the like is activated to supply gas from a gas supply source (not shown) into the first tank 10. The treated liquid in the first tank 10, which has been treated by the adsorbent treatment method using an adsorbent, is pressurized, and the treated liquid is forced into the inside of the hollow fiber membrane 30 for filtration in the first filtration step. In this disclosure, the entire process of filtering the liquid in the first tank 10 with the hollow fiber membrane 30 and sending it to the second tank 20 is referred to as the first filtration process. The gas for pressurization is supplied through the gas supply line 52 equipped with a pressure gauge 51 and the first tank gas supply line 53 by switching the three-way valve 61. At this time, the on / off valve 46 and the on / off valve 62 of the first gas vent line 55 are kept closed, and the on / off valve 63 of the second gas vent line 56 is kept open. The gas used can be selected from inert gases such as nitrogen, argon, and helium, as well as carbon dioxide and clean air filtered by a HEPA filter or similar device. The filtered permeate is stored in permeate tank 35, and the concentrated solution containing extracellular vesicles (first concentrated solution) is sent to the second tank 20. When the first concentrated liquid is present in the second tank 20, there is a space at the top of the second tank 20 where the first concentrated liquid is not present.

[0023] The hollow fiber membrane 30 preferably has an inner diameter of 0.2 mm to 1.4 mm, more preferably 0.2 mm to 1.0 mm, and even more preferably 0.4 mm to 1.0 mm. The hollow fiber membrane 30 is preferably an ultrafiltration membrane with a molecular weight cutoff of 100,000 to 1,000,000, more preferably an ultrafiltration membrane with a molecular weight cutoff of 100,000 to 800,000, and even more preferably an ultrafiltration membrane with a molecular weight cutoff of 100,000 to 600,000. The molecular weight cutoff is evaluated by the % permeability of gamma globulin when a 100 mg / L solution of gamma globulin (SIGMA bovine serum gamma globulin, molecular weight 150,000) in phosphate buffer is cross-flow permeated through a hollow fiber membrane 30 at a filtration pressure of 0.1 MPa (membrane velocity: 0.2 m / s) ((gamma globulin concentration in permeate / gamma globulin concentration in solution (100 mg / L) × 100). The hollow fiber membrane 30 preferably has a gamma globulin permeability of 5% to 95%, more preferably 10% to 80%, and even more preferably 10% to 70%. The hollow fiber membrane 30 may be a hydrophobic membrane such as a polyethersulfone membrane, or a cellulose-based hydrophilic membrane, but a cellulose-based hydrophilic membrane is preferred. Examples of cellulose-based hydrophilic membranes include cellulose acetate membranes, regenerated cellulose membranes, cellulose propionate membranes, cellulose butyrate membranes, and cellulose benzoate membranes. The hollow fiber membrane 30 can be made from Daisen Membrane Systems Co., Ltd.'s FUS5082 (polyethersulfone membrane; molecular weight cutoff 500,000, γ-globulin permeability 70%), Daisen Membrane Systems Co., Ltd.'s FUC1582 (cellulose acetate membrane; molecular weight cutoff 150,000, γ-globulin permeability 10%), etc.

[0024] The hollow fiber membrane 30 is positioned to connect the liquid inlet / outlet 10a of the first tank 10 and the liquid inlet / outlet 20a of the second tank 20. The hollow fiber membrane 30 is connected to the liquid inlet / outlet 10a of the first tank 10 by, for example, fitting the open end of the hollow fiber membrane 30 into a thin tube, such as a hypodermic needle, fixed to the liquid inlet / outlet 10a side of the first tank 10. The connection between the hollow fiber membrane 30 and the liquid inlet / outlet 20a of the second tank 20 can be carried out in a similar manner. The permeate tank 35 is for storing the permeate obtained by filtration in the hollow fiber membrane 30. In Figure 1, the permeate tank 35 is shown as a small object, but it can also be made into a larger tank that can accommodate most of the hollow fiber membrane 30.

[0025] In Figure 1, one hollow fiber membrane 30 is shown, but there may be multiple membranes; for example, it can be used as a bundle of 2 to 150 hollow fiber membranes. Alternatively, a hollow fiber membrane module may be formed in which multiple hollow fiber membranes (hollow fiber membrane bundles) 30 are housed in a case housing having multiple liquid inlets and outlets. When used as a hollow fiber membrane bundle, one end or both ends can be integrated with an adhesive. When using the hollow fiber membrane module, the multiple liquid inlets and outlets of the hollow fiber membrane module are connected to the liquid inlet and outlet 10a of the first tank 10 and the liquid inlet and outlet 20a of the second tank 20, and the liquid permeate outlet of the hollow fiber membrane module is connected to the permeate tank 35.

[0026] In the third step of filtration, it is preferable to filter with a membrane surface velocity in the range of 0.3 m / sec to 2 m / sec, and more preferably with a membrane surface velocity in the range of 0.5 m / sec to 1.5 m / sec. If the membrane velocity falls below 0.3 m / sec, the purification efficiency decreases. Conversely, if it exceeds 2 m / sec, the pressure level required to increase the membrane velocity becomes too high, resulting in excessive shear force applied to the extracellular vesicles during filtration. This can potentially alter the extracellular vesicles, which is undesirable. A method for maintaining the membrane surface velocity within the aforementioned range is to adjust the pressure at the inlet of the hollow fiber membrane 30 (on the liquid inlet / outlet 10a side of the first tank 10) to 0.01 MPa to 0.2 MPa, more preferably to 0.02 MPa to 0.15 MPa, and even more preferably to 0.03 MPa to 0.12 MPa. A method for maintaining the membrane surface velocity within the range is to adjust the pressure at the outlet of the hollow fiber membrane 30 (on the liquid inlet / outlet side 20a of the second tank 20) ​​to 0.03 MPa or less, more preferably to 0.01 MPa or less, and even more preferably to 0 MPa.

[0027] In the fourth step, a pump (not shown) is activated to supply gas from a gas supply source (not shown) into the second tank 20, pressurizing the liquid containing extracellular vesicles (first concentrate) in the second tank 20, and a second filtration step is performed in which the liquid containing extracellular vesicles is filtered by passing it through the inside of the hollow fiber membrane 30. In this disclosure, the entire process of filtering the liquid in the second tank 20 with the hollow fiber membrane 30 and sending it to the first tank 10 is referred to as the second filtration process. The filtered permeate is stored in permeate tank 35, and the concentrated solution containing extracellular vesicles (second concentrated solution) is sent to tank 10.

[0028] Although the second tank 20 is cylindrical in shape in Figure 1, it is not limited to this, and its shape and volume can be determined according to the conditions of the installation site and the processing volume. The second tank 20 is preferably transparent so that the liquid level inside can be observed visually, and is also preferably water-repellent to prevent liquid from adhering to and remaining on the inner wall surface of the second tank 20. The second tank 20 is preferably made of part or all of an acrylic resin such as polyacrylonitrile or polyacrylic acid ester, polycarbonate, or fluororesin. It is preferable that the first tank 10 and the second tank 20 have the same shape and the same volume. The first tank 10 and the second tank 20 are positioned at the same height, with a gap between them.

[0029] The gas for pressurization is supplied through the gas supply line 52 and the second tank gas supply line 54 by switching the three-way valve 61. At this time, the on / off valves 63 and 46 of the second gas vent line 56 are kept closed, and the on / off valve 62 of the first gas vent line 55 is kept open. The gas used can be selected from inert gases such as nitrogen, argon, and helium, as well as carbon dioxide and clean air filtered by a HEPA filter or similar device.

[0030] It is preferable that the film surface velocity in the fourth step be within the same range as the film surface velocity in the third step. In the fourth step, the inlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 20a side of the second tank 20) ​​is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably to 0.02 MPa to 0.15 MPa, and even more preferably to 0.03 MPa to 0.12 MPa. In the fourth step, the outlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet side 10a of the first tank 10) is preferably adjusted to 0.03 MPa or less, more preferably to 0.01 MPa or less, and even more preferably to 0 MPa. The third and fourth steps can be carried out continuously by switching the three-way valve 61 while continuously supplying gas from the gas supply source through the gas supply line 52.

[0031] Subsequently, the extracellular vesicles in the liquid containing the extracellular vesicles are separated and purified by repeating the first filtration step (third step) and the second filtration step (fourth step) multiple times. When the first and second filtration steps are repeated multiple times, it is preferable to increase the dilution factor of the buffer solution to be filtered in the first tank 10 of the first filtration step (i.e., the dilution factor of the solution to be filtered in the first filtration step) as the number of repetitions increases. For example, it can be increased in the range of 2 to 15 times the volume, and preferably in the range of 2 to 10 times the volume. By performing the first and second filtration steps alternately in this alternating tangential flow filtration, a concentrated solution with an increased concentration of extracellular vesicles can be obtained.

[0032] The method for separating and purifying extracellular vesicles according to this disclosure is preferably carried out in such a way that, when the starting material solution containing extracellular vesicles and waste products is the culture supernatant of mesenchymal stem cells containing extracellular vesicles, the amount of protein contained in the culture supernatant is reduced to 1 / 5 or less, preferably 1 / 10 or less.

[0033] In the method for separating and purifying extracellular vesicles according to this disclosure, when the solution containing extracellular vesicles and waste products is the culture supernatant of mesenchymal stem cells containing extracellular vesicles, it is preferable that the amount of extracellular vesicles contained in the concentrated solution obtained by alternately performing the first filtration step and the second filtration step is such that the concentration ratio is 5 times or more and the recovery rate is 50% or more, based on the amount of extracellular vesicles contained in the culture supernatant.

[0034] Each aspect disclosed herein can be combined with any other features disclosed herein. The configurations and combinations thereof in each embodiment are examples only, and additions, omissions, substitutions, and other modifications can be made as appropriate without departing from the spirit of the disclosure of the present invention. This disclosure is not limited by the embodiments, but is limited only by the claims. [Examples]

[0035] Example 1 (Example of adsorbent treatment method) <Preparation of culture supernatant of mesenchymal stem cells containing extracellular vesicles> As a solution containing extracellular vesicles and waste products, we used the culture supernatant of mesenchymal stem cells containing extracellular vesicles, prepared by the method described below. Human adipose tissue-derived mesenchymal stem cells were cultured using Ultra ExoMCulture Medium for Extracellular Vesicles (FK-K0204024, Santeja). Using a 15cm dish, the cells covered approximately 80% of the surface of the culture vessel's adhesive surface. The culture medium was then replaced with phenol red-free Ultra ExoM Culture Medium and incubated for 48 hours. Subsequently, the culture supernatant was centrifuged at 2000 × g for 10 minutes at 4°C to remove cell debris, and the culture supernatant was prepared. Quantitative analysis of the protein concentration and CD63-positive extracellular vesicles in the culture supernatant was performed using the Bradford method and the ExoScreen method, respectively. The results showed a protein concentration of 1.1 mg / mL and an ExoScreen signal intensity of 6,092.

[0036] <Isolation and purification of extracellular vesicles from culture supernatant> To 25 mL of the culture supernatant, 2.5 mL of the adsorbent carrier CaptoCore700 (17548101, Cytiva), which had been equilibrated with 50 mM Tris-HCl (pH 7.2) solution, was added, and the mixture was inverted and mixed at a rotation speed of 10 r / min at room temperature (approximately 20°C) for 15 minutes using a rotator. Subsequently, the adsorbent was removed from the carrier-cell supernatant mixture using a Stericup 0.22 μm filtration unit (S2GPU11RE, MerckMillipore). Quantitative analysis of the protein concentration and extracellular vesicles in the culture supernatant treated with the adsorbent revealed a protein concentration of 0.4 mg / mL and an ExoScreen signal intensity of 6,075.

[0037] Example 2 (Implementation of adsorbent treatment method and membrane separation method) In Example 1, the treated solution obtained by the adsorbent treatment method was used, and a membrane separation method was performed using the separation and purification apparatus 1 shown in Figure 1. The separation and purification was carried out at room temperature (approximately 20°C). (Separation and purification apparatus 1 shown in Figure 1) • First tank 10 and second tank 20 Material: Acrylic ester resin Size: Length 25cm, Inner diameter 0.25cm, Capacity 120cm 3 • 40 buffer tanks Capacity: 1.6L • Hollow fiber membrane 30 Inner diameter 0.8mm, outer diameter 1.3mm, length 30cm, membrane area 7.5cm 2 , Hollow fiber membrane made of cellulose acetate (CA) with a fractional molecular weight of 300,000 (Product name: Prototype FUC3081exp., manufactured by Daisen Membrane Systems Co., Ltd.)

[0038] <Implementation of a method for separating and purifying extracellular vesicles from a treatment solution using an adsorbent treatment method> Using the separation and purification apparatus shown in Figure 1, extracellular vesicles were separated and purified in the following steps. (1) The treated liquid from the adsorbent treatment method was placed into the first tank 10. (2) Nitrogen gas was supplied to the upper space of the first tank 10 at a pressure of 0.05 MPa, and tangential flow filtration was performed while passing the diluted solution of the adsorbent treatment method described above through the inside of the hollow fiber membrane 30. At this time, the second tank 20 was open to the atmosphere with the on / off valve 56 open, and the pressure was zero. Also, the linear velocity of the membrane flowing inside the hollow fiber membrane 30 was 1.0 m / s. The linear velocity of the membrane was calculated from the rate of increase in the amount of concentrated liquid in the second tank 20. The permeate was stored in the permeate tank 35, and the concentrated liquid (first concentrated liquid) was transferred to the second tank 20 (first filtration step). (3) When the diluted solution of the adsorbent treatment method in the first tank 10 has passed through the inside of the hollow fiber membrane 30 and been filtered, and most of it has moved to the second tank 20, nitrogen gas is supplied to the second tank 20 by switching the three-way valve 61, and at the same time the pressure in the first tank 10 is released by opening the on / off valve 62. (4) Through this operation, the first concentrate is filtered while being transferred from the second tank 20 to the first tank 10, the permeate is stored in the permeate tank 35, and the concentrate (second concentrate) is transferred into the first tank 10 (second filtration step). When most of the first concentrated liquid in the second tank 20 has moved to the first tank 10, the three-way valve 61 is switched to filter the second concentrated liquid in the first tank 10 again through the hollow fiber membrane 30, and the concentrated liquid is transferred back into the second tank 20 (first filtration step). Alternating tangential flow filtration was performed, repeating the same first and second filtration processes multiple times. Alternating tangential flow filtration was performed until the liquid volume reached 1.5 ml, yielding a concentrated solution (final concentrate) with increased exosome concentration.

[0039] When the exosomes in the final concentrate were quantitatively evaluated using the ExoScreen method, their signal intensity was 92,500. The signal intensity of exosomes in the initial 25.0 ml of culture supernatant was 6,075. By separating and purifying them using a hollow fiber membrane with a molecular weight cutoff of 300,000 (equivalent pore size of 10-20 nm), the concentration of exosomes in the final 1.5 ml concentrate was approximately 15 times. The recovery rate, calculated from ExoScreen signal intensity and solution volume, was 91%. The residual protein concentration was approximately 0.2 mg / mL, representing about 18% of the culture supernatant (protein concentration 1.1 mg / mL).

[0040] Comparative Example 1 Using the culture supernatant of mesenchymal stem cells containing extracellular vesicles prepared in Example 1, the membrane separation method of Example 2 was applied directly without applying the adsorbent treatment method to obtain a final concentrate of 1.2 mL. The residual protein concentration was 0.7 mg / mL, which was more than three times higher than in the examples. During the alternating tangential flow filtration process, the filtrate volume was sampled over time, and the filtration volume was calculated from the change in mass. As a result, in Comparative Example 1, the cumulative permeate volume was 2.5 cm³. 3 / cm 2 The filtration time to reach this point was 45 minutes. In Example 2, obtained in the same manner, the cumulative permeate volume was 2.5 cm³. 3 / cm 2 The filtration time to reach the desired result was reduced to 22 minutes, less than half the original time. [Industrial applicability]

[0041] The separation and purification method described herein can be used when separating and purifying extracellular vesicles from a culture medium. [Explanation of Symbols]

[0042] 1 Separation and purification equipment 10 Tank No. 1 20 Tank No. 2 30 Hollow fiber membrane 35 Permeate Tank 40 buffer tanks

Claims

1. A method for separating and purifying extracellular vesicles from a solution containing extracellular vesicles and waste products, The separation and purification method is a method that combines an adsorbent treatment method, in which an adsorbent is brought into contact with a solution containing the extracellular vesicles and unwanted materials, thereby adsorbing and retaining the unwanted materials (excluding the extracellular vesicles) on the adsorbent, with a membrane separation method. The adsorbent is a porous granular material in which the inside of the pores is positively charged, and is capable of adsorbing and retaining negatively charged unwanted substances, excluding the extracellular vesicles, within the pores. The filtration method in the aforementioned membrane separation method is The hollow fiber membrane used in the aforementioned membrane separation method has an inner diameter of 0.2 mm to 1.4 mm and a fractional molecular weight of 100,000 to 1,000,000. A first filtration step involves pressurizing the inlet pressure at one end of the hollow fiber membrane to 0.01 MPa to 0.2 MPa, thereby injecting the treated liquid from the adsorbent treatment method or the second concentrated liquid obtained in the second filtration step into the first opening at one end of the hollow fiber membrane and filtering it to separate it into a permeate and a first concentrated liquid. The system includes a second filtration step in which the inlet pressure at the other end of the hollow fiber membrane is increased to 0.01 MPa to 0.2 MPa, thereby forcing the first concentrated liquid into the second opening at the other end of the hollow fiber membrane for filtration and separating it into a permeate and a second concentrated liquid. This method involves performing the first filtration step and the second filtration step multiple times alternately to obtain a concentrated solution in which the concentration of extracellular vesicles is increased by alternating tangential flow filtration. A method for separating and purifying extracellular vesicles, wherein the membrane surface velocity in the first and second filtration steps is 0.3 m / sec to 2 m / sec.

2. The method for separating and purifying extracellular vesicles according to claim 1, wherein the solution containing the extracellular vesicles and waste products is a culture supernatant containing extracellular vesicles.

3. The method for separating and purifying extracellular vesicles according to claim 1, wherein the solution containing the extracellular vesicles and waste products is the culture supernatant of mesenchymal stem cells containing extracellular vesicles.

4. The separation and purification method according to any one of claims 1 to 3, wherein the adsorbent treatment method is selected from a method of passing a solution containing the extracellular vesicles and unwanted materials through a column packed with the adsorbent, or a method of adding the adsorbent to a container containing the solution containing the extracellular vesicles and unwanted materials, and then stirring and mixing it.

5. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 4, wherein the method involves performing multiple adsorbent treatments using multiple adsorbents having different adsorption properties.

6. The method for separating and purifying extracellular vesicles according to claim 5, wherein the plurality of adsorbents having different adsorption performance are selected from one or more of those having different pore diameters of porous granules, different specific surface areas of porous granules, different average particle diameters of porous granules, different types of substrates constituting the porous granules, and different types and densities of functional groups present on the inner surface of the pores of porous granules.

7. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 6, wherein the separation and purification method is a method of alternately performing the adsorbent treatment method and the membrane separation method, or a method of performing them randomly, and the first treatment is the adsorbent treatment method.

8. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 7, wherein the filtration method in the membrane separation method is a step of filtering the treated solution from the adsorbent treatment method using a microfiltration membrane with a pore size of 0.1 μm to 0.5 μm, and then using the filtrate from the microfiltration membrane, performing the first filtration step and the second filtration step alternately multiple times.

9. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 8, wherein when the first filtration step is performed, a buffer solution is added to the treated solution from the adsorbent treatment method or the second concentrated solution obtained in the second filtration step to dilute it, and then the first filtration step is performed.

10. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 9, wherein the first filtration step and the second filtration step are carried out by introducing a gas selected from nitrogen gas, an inert gas, carbon dioxide, and air filtered by a HEPA filter under pressure.

11. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 10, wherein the amount of protein contained in a solution containing the extracellular vesicles and waste materials that serve as the starting material is reduced to 1 / 5 or less.

12. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 11, wherein, when the amount of extracellular vesicles contained in the solution containing the extracellular vesicles and waste products is used as a reference, the amount of extracellular vesicles contained in the concentrate measured by the ExoScreen method is such that the concentration ratio is 5 times or more and the recovery rate is 50% or more.

13. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 12, wherein the hollow fiber membrane is a hollow fiber membrane module in which a plurality of hollow fiber membranes are housed in a case housing having a plurality of liquid inlets and outlets.

14. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 13, wherein when the first filtration step and the second filtration step are performed alternately, the dilution factor of the material to be filtered in the first filtration step is increased as the number of executions increases.

15. A method for separating and purifying extracellular vesicles according to any one of claims 1 to 14, wherein when the first filtration step and the second filtration step are performed alternately, the dilution ratio of the material to be filtered in the first filtration step is increased in the range of 2 to 15 times the volume as the number of times the steps are performed increases.

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