Method for isolating and purifying extracellular vesicles

Abstract

To provide a method for separating and purifying extracellular vesicles.SOLUTION: Wherein the hollow fiber membrane has an inner diameter of 0.2 mm to 1.4 mm and a molecular weight cut-off of 100,000 to 1,000,000, and the filtration method comprises a first filtration step of pressurizing and filtering a culture supernatant of mesenchymal stem cells through a first opening on one end side of the hollow fiber membrane to separate the culture supernatant into a permeated liquid and a first concentrated liquid, and a second filtration step of pressurizing and filtering the first concentrated liquid through a second opening on the other end side of the hollow fiber membrane; A method for separating and purifying extracellular vesicles, the method comprising a second filtration step of separating the extracellular vesicles into a permeate and a second concentrate, wherein a concentrate having an increased concentration of the extracellular vesicles is obtained by alternate tangential flow filtration in which the first filtration step and the second filtration step are alternately performed a plurality of times, and a membrane surface speed in the first filtration step and the second filtration step is 0.3 to 2m / sec.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] As a method for separating and purifying useful substances from a culture medium, a method using a separation membrane is known. Patent Document 1 describes an invention of a method for concentrating a culture solution of unicellular algae, in which the culture solution is concentrated by cross-flow filtration using a hollow fiber ultrafiltration membrane (UF membrane) module with a molecular weight cutoff of 10,000 to 1,000,000, and periodic underflow washing is performed. When a culture solution is concentrated by cross-flow filtration, the concentrate exists on the outside of the UF membrane and the permeate enters the inside of the UF membrane. When underflow washing is performed, the washing water enters the inside of the UF membrane and then comes out on the outside of the UF membrane, thereby cleaning the UF membrane.

[0003] Patent Document 2 describes an invention of a method for recovering useful substances, which includes a bleeding step of discharging a culture medium from a cell culture tank and adding a fresh medium in an amount equal to the amount of the discharged culture medium to the culture tank, and a filtration step of filtering the culture medium extracted from the culture tank using a porous membrane that does not substantially have a dense layer, wherein 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. It is stated that the useful substance is selected from the group consisting of proteins, viruses, exosomes, and nucleic acids. It is stated that the tangential flow filtration can also be carried out as an alternating tangential flow filtration. Extracellular vesicles are a collective term for lipid bilayer-covered particles without a nucleus that are released outside of cells. They contain nucleic acids, proteins, lipids, and various metabolic products, and include exosomes, microvesicles, and apoptotic vesicles.

[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 Application Publication No. 2018-76291 [Non-patent literature]

[0006] [Non-Patent Document 1] Cytometry Research 26(1):1~6, 2016, "New Developments in Liquid Biopsy Using Exosomes", Yusuke Yoshioka, Takahiro Ochiya Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present disclosure is to provide a method for separating and purifying extracellular vesicles. [Means for solving the problem]

[0008] The present disclosure provides a method for separating and purifying extracellular vesicles, which comprises filtering a culture supernatant of mesenchymal stem cells containing extracellular vesicles through a hollow fiber membrane, The hollow fiber membrane 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 comprises: a first filtration step in which the mesenchymal stem cell culture supernatant is pressure-filtered through a first opening at one end of the hollow fiber membrane to separate the supernatant into a permeate and a first concentrate; a second filtration step in which the first concentrated liquid is pressure-injected through a second opening at the other end of the hollow fiber membrane to be filtered and separated into a permeate and a second concentrated liquid; A method for obtaining a concentrated solution having an increased concentration of extracellular vesicles by alternating tangential flow filtration, in which the first filtration step and the second filtration step are alternately performed multiple times, The method for separating and purifying extracellular vesicles is provided, wherein the membrane surface velocity in the first filtration step and the second filtration step is 0.3 m / sec to 2 m / sec. [Effects of the Invention]

[0009] According to the method for separating and purifying extracellular vesicles disclosed herein, extracellular vesicles can be concentrated to a high concentration. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram showing a separation and purification apparatus for carrying out a method for separating and purifying extracellular vesicles. [Figure 2] 2 is a partially enlarged view of a separation and purification device of an embodiment different from that of FIG. 1, which is used in the separation and purification flow shown in FIG. [Figure 3] This is a chart showing the measurement of extracellular vesicles (exosomes) in the culture supernatant used in the Examples using the NanoSight method. [Figure 4] This is a chart showing the measurement of extracellular vesicles (exosomes) in the final concentrated solution of the example using the NanoSight method. DETAILED DESCRIPTION OF THE INVENTION

[0011] One embodiment of the method for separating and purifying extracellular vesicles will be described using a production flow using the separation and purification apparatus 1 shown in FIG. In the first step, a culture supernatant of mesenchymal stem cells containing extracellular vesicles is placed in a first tank 10. 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. Culturing methods thereof are described, for example, in JP 2011-67175 A and JP 2003-52360 A.

[0012] Although the first tank 10 is cylindrical in shape in FIG. 1, it is not limited to this, and the shape and volume can be determined depending on the conditions of the installation location and the amount of treatment. The first tank 10 is preferably transparent so that the liquid level inside can be visually observed, and furthermore, it is 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 in part or in whole from an acrylic resin such as polyacrylonitrile or polyacrylic ester, polycarbonate, or fluororesin. The first tank 10 may be provided with a withdrawal line for withdrawing the final concentrate from within the first tank 10.

[0013] As shown in Figure 2, the connection portion 11 with the hollow fiber membrane 30, which includes the liquid inlet / outlet 10a of the first tank 10, has a conical inclined surface 11a whose diameter decreases from the first tank 10 side to the hollow fiber membrane 30 side, and a cylindrical vertical surface 11b. In FIG. 2, the connecting portion 11 has a conical inclined surface 11a and a cylindrical vertical surface 11b, but as long as it has the conical inclined surface 11a, the cylindrical vertical surface 11b does not have to be provided. The presence of the connection part 11 shown in Figure 2 is preferable because it prevents the liquid containing extracellular vesicles or a concentrated solution thereof from accumulating at the bottom side of the first tank 10, thereby increasing the recovery rate of extracellular vesicles.

[0014] Before the first step, a pretreatment step using a microfiltration membrane (microfiltration membrane module) can be carried out as necessary. The microfiltration membrane (microfiltration membrane module) preferably has a pore size of 0.1 μm to 0.5 μm. In the pretreatment step, the filtrate (pretreatment liquid) obtained by filtering the liquid containing extracellular vesicles through a microfiltration membrane (microfiltration membrane module) is sent to the first tank 10.

[0015] In the second step, with the opening / closing valve (such as an electromagnetic valve) 46 open, the buffer solution in the buffer solution tank 40 is supplied from the buffer solution supply line 45 into the first tank 10 to dilute the liquid (or pretreatment liquid) containing extracellular vesicles. When the first tank 10 contains a diluted solution of mesenchymal stem cell culture supernatant (or pretreatment solution) containing extracellular vesicles, there remains space above the first tank 10 where the diluted solution is not present. The buffer solution in the buffer solution tank 40 is preferably a medical or biochemical buffer solution, and examples of buffer solutions that can be used include phosphate buffer solution (PBS), Tris-HCl buffer solution, sodium citrate buffer solution, citrate-phosphate buffer solution, acetate buffer solution, and borate buffer solution. When the buffer tank 40 is replenished with the buffer, it is replenished through a buffer refill line 41 . In addition, the order of the first and second steps can be reversed, and the buffer solution in the buffer tank 40 can be supplied from the buffer solution supply line 45 into the first tank 10, and then the mesenchymal stem cell culture supernatant (or pretreatment solution) containing extracellular vesicles can be placed into the first tank 10. Alternatively, the first and second steps can be combined into one step, and a diluted solution can be prepared by adding and mixing a culture supernatant (or pretreatment solution) of mesenchymal stem cells containing extracellular vesicles with a buffer solution in a separately provided mixing tank, and then placing the diluted solution in the first tank 10.

[0016] In the third step, a pump or the like (not shown) is operated to supply gas from a gas supply source (not shown) into the first tank 10, and the mesenchymal stem cell culture supernatant (or pretreatment liquid) containing extracellular vesicles in the first tank 10 is pressurized, thereby performing the first filtration step of forcing the mesenchymal stem cell culture supernatant (or pretreatment liquid) containing extracellular vesicles into the inside of the hollow fiber membrane 30 and filtering it. In the present disclosure, the 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 sent through a gas supply line 52 equipped with a pressure gauge 51 and a first tank gas supply line 53 by switching a 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 closed, and the on-off valve 63 of the second gas vent line 56 is open. The gas that can be used may be selected from inert gases such as nitrogen gas, argon, and helium, carbon dioxide, and clean air filtered with a HEPA filter or the like. The filtered permeate is stored in the permeate tank 35, and the concentrate containing extracellular vesicles (first concentrate) is sent to the second tank 20. When the second tank 20 contains the first concentrated liquid, a space remains above the second tank 20 where the first concentrated liquid does not exist.

[0017] 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 200,000 to 800,000, and even more preferably an ultrafiltration membrane with a molecular weight cutoff of 300,000 to 600,000. The molecular weight cutoff is evaluated by the γ-globulin permeability (%) ((γ-globulin concentration in permeate / γ-globulin concentration in solution (100 mg / L) × 100) when a 100 mg / L solution of γ-globulin (bovine serum γ-globulin manufactured by SIGMA, molecular weight 150,000) in a phosphate buffer solution is passed through the hollow fiber membrane 30 at a filtration pressure of 0.1 MPa (membrane surface velocity: 0.2 m / s). The hollow fiber membrane 30 has a γ-globulin permeability of 5% to 95%, more preferably 10 to 80%, and even more preferably 30 to 70%. The hollow fiber membrane 30 may be a hydrophobic membrane such as a polyethersulfone membrane or a hydrophilic cellulose membrane, but a hydrophilic cellulose membrane is preferred. Examples of hydrophilic cellulose membranes include cellulose acetate membrane, regenerated cellulose membrane, cellulose propionate membrane, cellulose butyrate membrane, and cellulose benzoate membrane. The hollow fiber membrane 30 may be FUS5082 (polyethersulfone membrane; molecular weight cutoff 500,000, gamma globulin permeability 70%) manufactured by Daisen Membrane Systems Co., Ltd., or FUC1582 (cellulose acetate membrane; molecular weight cutoff 150,000, gamma globulin permeability 10%) manufactured by Daisen Membrane Systems Co., Ltd.

[0018] The hollow fiber membrane 30 is arranged to connect the liquid inlet / outlet 10 a of the first tank 10 and the liquid inlet / outlet 20 a of the second tank 20 . The hollow fiber membrane 30 is connected to the liquid inlet / outlet 10a of the first tank 10, for example, by fitting the open end of the hollow fiber membrane 30 into a thin tube such as a syringe needle fixed to the liquid inlet / outlet 10a side of the first tank 10. The hollow fiber membrane 30 can also be connected to the liquid inlet / outlet 20a of the second tank 20 in a similar manner. The permeate tank 35 is for storing the permeate obtained by filtration through the hollow fiber membrane 30 . In FIG. 1, the permeate tank 35 is shown small, but it can also be a large tank that can accommodate most of the hollow fiber membranes 30.

[0019] Although one hollow fiber membrane 30 is shown in FIG. 1, a plurality of hollow fiber membranes may be used, and a bundle of, for example, 2 to 150 hollow fiber membranes may be used. Alternatively, the hollow fiber membrane module may be one in which a plurality of hollow fiber membranes (hollow fiber membrane bundle) 30 are housed in a case housing having a plurality of liquid inlets and outlets. When used as a hollow fiber membrane bundle, one or both ends can be bonded together with an adhesive. When the hollow fiber membrane module is used, the multiple liquid inlets and outlets of the hollow fiber membrane module are connected to the liquid inlets and outlets 10a of the first tank 10 and the liquid inlets and outlets 20a of the second tank 20, and the liquid permeation outlet of the hollow fiber membrane module is further connected to the permeate tank 35.

[0020] The filtration in the third step is preferably carried out at a membrane velocity in the range of 0.3 m / sec to 2 m / sec, more preferably 0.5 m / sec to 1.5 m / sec. If the membrane velocity is 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, and the shear force applied to the extracellular vesicles during filtration also becomes too high, which may result in denaturation of the extracellular vesicles. The method for maintaining the membrane surface velocity within the above range is to adjust the inlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 10a side of the first tank 10) to preferably 0.01 MPa to 0.2 MPa, more preferably 0.02 MPa to 0.15 MPa, and even more preferably 0.03 MPa to 0.12 MPa. The method for maintaining the membrane surface velocity within the above range is preferably to adjust the outlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 20a side 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.

[0021] In the fourth step, a pump or the like (not shown) is operated to supply gas from a gas supply source (not shown) into the second tank 20, pressurizing the liquid (first concentrated liquid) containing extracellular vesicles in the second tank 20, and performing a second filtration step in which the liquid containing extracellular vesicles is passed through the inside of the hollow fiber membrane 30 and filtered. In the present disclosure, the 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 the permeate tank 35, and the concentrate (second concentrate) containing extracellular vesicles is sent to the first tank 10.

[0022] Although the second tank 20 is cylindrical in shape in FIG. 1, it is not limited to this, and the shape and volume can be determined depending on the conditions of the installation location and the amount of treatment. 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 partially or entirely of an acrylic resin such as polyacrylonitrile or polyacrylic ester, polycarbonate, or fluororesin. The first tank 10 and the second tank 20 preferably have the same shape and the same volume. The first tank 10 and the second tank 20 are arranged at the same height with a gap between them.

[0023] The gas for pressurization is sent 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 valve 63 of the second gas vent line 56 and the on-off valve 46 are closed, and the on-off valve 62 of the first gas vent line 55 is open. The gas that can be used may be selected from inert gases such as nitrogen gas, argon, and helium, carbon dioxide, and clean air filtered with a HEPA filter or the like.

[0024] The surface velocity in the fourth step is preferably in the same range as the surface velocity in the third step. The inlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 20a side of the second tank 20) ​​in the fourth step is preferably adjusted to 0.01 MPa to 0.2 MPa, more preferably 0.02 MPa to 0.15 MPa, and even more preferably 0.03 MPa to 0.12 MPa. The outlet pressure of the hollow fiber membrane 30 (on the liquid inlet / outlet 10a side of the first tank 10) in the fourth step is preferably adjusted to 0.03 MPa or less, more preferably adjusted to 0.01 MPa or less, and even more preferably adjusted 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.

[0025] Thereafter, the first filtration step (third step) and the second filtration step (fourth step) are repeated multiple times to separate and purify the extracellular vesicles in the liquid containing the extracellular vesicles. When the first filtration step and the second filtration step 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 by volume, and preferably it can be increased in the range of 2 to 10 times by volume. In this way, by alternately performing the first filtration step and the second filtration step, a concentrated solution with an increased concentration of extracellular vesicles can be obtained.

[0026] The method for separating and purifying extracellular vesicles of the present disclosure is preferably carried out so as to reduce the amount of protein contained in the culture supernatant of mesenchymal stem cells containing extracellular vesicles, which serves as the starting material, to 1 / 5 or less, preferably 1 / 10 or less. Furthermore, it is preferable to reduce the insulin concentration in the separated and purified concentrate to 5 mg / L or less, preferably 2 mg / L or less, and more preferably 1 mg / L or less.

[0027] The method for separating and purifying extracellular vesicles disclosed herein is preferably carried out so that, based on the amount of extracellular vesicles contained in the culture supernatant of mesenchymal stem cells as the starting material, the amount of extracellular vesicles contained in the concentrate obtained by alternately repeating the first filtration step and the second filtration step (the amount measured using the ExoScreen method described in Non-Patent Document 1) is concentrated at a rate of 5 times or more and the recovery rate is 50% or more.

[0028] Each feature disclosed herein may be combined with any other feature disclosed herein. The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate within the scope of the gist of the disclosure of the present invention. The present disclosure is not limited by the embodiments, but is limited only by the claims. [Example]

[0029] Example 1 (Separation and purification device 1 shown in Figure 1) First tank 10 and second tank 20 Material: Polyacrylonitrile Size: Length 25cm, inner diameter 0.25cm, capacity 120cm 3 Buffer tank 40 Capacity: 1.6L Hollow fiber membrane 30 Inner diameter 0.8mm, outer diameter 1.3mm, length 50cm, membrane area 12.6cm 2 Polyethersulfone (PES) hollow fiber membrane with a molecular weight cutoff of 500,000 (product name FUS5081, manufactured by Daisen Membrane Systems Co., Ltd.)

[0030] (Preparation of culture supernatant) Human adipose-derived mesenchymal stem cells were used as the mesenchymal stem cells, and Ultra ExoM Culture Medium for Extracellular Vesicles (product number FK-K0204024, manufactured by Santeja Co., Ltd.) was used as the culture medium. Mesenchymal stem cells were cultured in a 15 cm dish containing medium, and when the mesenchymal stem cells covered approximately 80% of the adhesive surface of the culture vessel, the medium was changed to Ultra ExoM Culture Medium, which does not contain phenol red, and the cells were cultured for 48 hours. Thereafter, the culture supernatant was centrifuged at 2000×g for 10 minutes at 4° C. to remove cell debris, and a culture supernatant was prepared.

[0031] (Quantitative evaluation of culture supernatant) When the extracellular vesicles in the culture supernatant were quantitatively evaluated by the ExoScreen method, the signal intensity was 30,914. In addition, the extracellular vesicles in the culture supernatant were quantitatively evaluated using the NanoSight method (product name NanoSight NS300, manufactured by Malvern) with a 405 nm wavelength laser module, and the particle size distribution was found to have multiple peaks ranging from 50 nm to 600 nm (Figure 3). In addition, the number of extracellular vesicle particles measured by the NanoSight method was 0.3 × 10 9 The number was 1 / ml. Analysis of the proteins contained in the culture supernatant by SDS-PAGE (CBB staining) revealed that the protein contained was mainly a protein with a molecular weight of approximately 70,000. Quantitative analysis by the Bradford method revealed that the total amount of protein in 25.0 ml of culture supernatant was 29.2 mg (protein concentration: 1.2 mg / ml). Furthermore, ELISA quantification of insulin contained in the culture supernatant revealed a value of 375 μg.

[0032] <Implementation of a method for separating and purifying extracellular vesicles from culture supernatant containing extracellular vesicles> Extracellular vesicles were separated and purified from the culture supernatant containing extracellular vesicles using the separation and purification device shown in Figure 1. The separation and purification was carried out at room temperature (approximately 20°C). (1) The culture supernatant was completely filtered through a microfiltration membrane with a pore size of 0.22 μm (Millex-GP, made of PES, manufactured by Millipore Corporation) that can be attached to a syringe cylinder, to obtain a filtrate (pretreated liquid). (2) In a mixing vessel not shown in FIG. 1, 25 ml of the filtrate was mixed with 50 ml of phosphate buffer solution (PBS) to prepare 75 ml of diluted culture supernatant. (3) 75 ml of the diluted culture supernatant in the mixing vessel was placed in the first tank 10. (4) Nitrogen gas was supplied to the upper space of the first tank 10 at a pressure of 0.1 MPa, and the diluted culture supernatant was passed through the inside of the hollow fiber membrane 30 while performing tangential flow filtration. At this time, the second tank 20 was open to the atmosphere with the on-off valve 56 open, and the pressure was zero. The membrane surface linear velocity flowing inside the hollow fiber membrane 30 was 1.0 m / s. The membrane surface linear velocity was calculated from the rate of increase in the amount of concentrated liquid in the second tank 20. The permeated liquid was stored in the permeated liquid tank 35, and the concentrated liquid (first concentrated liquid) was transferred into the second tank 20 (first filtration step). (5) When the diluted culture supernatant in the first tank 10 was filtered through the inside of the hollow fiber membrane 30 and most of it transferred to the second tank 20, nitrogen gas was 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 was released by opening the opening / closing valve 62. (6) By this operation, the first concentrated liquid was filtered while being transferred from the second tank 20 to the first tank 10, the permeated liquid was stored in the permeated liquid tank 35, and the concentrated liquid (second concentrated liquid) was transferred into the first tank 10 (second filtration process). When most of the first concentrated liquid in the second tank 20 had transferred to the first tank 10, the three-way valve 61 was switched to filter the second concentrated liquid in the first tank 10 again using the hollow fiber membrane 30, and the concentrated liquid was transferred into the second tank 20 (first filtration process). Alternating tangential flow filtration was carried out by repeating the same first filtration step and second filtration step multiple times.

[0033] While repeating the first and second filtration steps (alternating tangential flow filtration) described above in (4) to (6), when the volume of the concentrated solution in the first tank 10 reached approximately 25 ml, 50 ml of phosphate buffer (calcium- and magnesium-free phosphate buffered saline) (10x PBS Buffer manufactured by Nippon Gene Co., Ltd.) from the buffer tank 40 was added. The gas phase (space not containing the buffer solution) of the buffer solution tank 40 was filled with nitrogen gas to compensate for the loss of the buffer solution.

[0034] The separation and purification steps (4) to (6) above (alternating tangential flow filtration) were repeated a total of four times, and finally, a total of 250 ml of phosphate buffer was added to the initial 25 ml of culture supernatant. After the fourth addition of phosphate buffer (75 ml of concentrated solution), the solution was filtered without dilution by alternating tangential flow filtration until the volume reached 2.1 ml, yielding a concentrated solution with an increased exosome concentration (final concentrated solution).

[0035] The exosomes in the final concentrate were quantitatively evaluated using the ExoScreen method, and the signal intensity was 233,323. The signal intensity of exosomes in the initial 25.0 ml of culture supernatant was 30,914, and by separating and purifying them using a hollow fiber membrane with a molecular weight cutoff of 500,000 (equivalent to a membrane pore size of 20 nm), the exosomes in the final concentrated solution (2.1 ml) were concentrated approximately 7.5 times. The recovery rate of exosomes was approximately 65% ​​((233,323 × 2.1 / 30,914 × 25.0) × 100). Furthermore, when the particle size distribution of the exosomes in the final concentrate was measured using the NanoSight method, it was found to have a peak at a particle size of 100 nm, as shown in Figure 4. Furthermore, the number of particles was 4.7 × 10 9 The number was 1 / ml. On the other hand, the total amount of protein in the final concentrated solution (2.1 ml) was 1.9 mg, and the amount of insulin was 1.8 μg (insulin concentration 0.9 mg / l). Compared to the initial culture supernatant (25 ml) containing exosomes (total amount of protein 29.2 mg, insulin amount 375 μg (insulin concentration 15 mg / l)), the total amount of protein was reduced to 6.5% and the amount of insulin to 0.5%. During the above alternating tangential flow filtration process, the amount of filtrate was sampled over time, and the filtration rate was calculated from the change in mass. 1 hour, membrane area 1m 2 The converted filtration rate per 0.1 MPa pressure drops significantly in the early stages, but after about 20 minutes of filtration, it reaches 270-300 (average 280) L / m 2 h, and separation and purification was completed approximately 65 minutes after the start of filtration. [Industrial Applicability]

[0036] The separation and purification method of the present disclosure can be used to separate and purify extracellular vesicles from culture medium. [Explanation of symbols]

[0037] 1 Separation and purification equipment 10. First Tank 20 Second Tank 30 Hollow fiber membrane 35 Permeate tank 40 Buffer Tank

Claims

1. A method for separating and purifying extracellular vesicles, comprising filtering a culture supernatant of mesenchymal stem cells containing extracellular vesicles through a hollow fiber membrane, the hollow fiber membrane 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 comprises: a first filtration step in which a culture supernatant of mesenchymal stem cells containing the extracellular vesicles is supplied and an inlet pressure at one end of the hollow fiber membrane is increased to 0.01 MPa to 0.2 MPa, thereby forcing the culture supernatant of mesenchymal stem cells through a first opening at one end of the hollow fiber membrane to filter the culture supernatant and separate it into a permeate and a first concentrate; a second filtration step in which the first concentrated liquid is supplied and an 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 a second opening at the other end of the hollow fiber membrane and filtering the liquid, thereby separating the liquid into a permeate liquid and a second concentrated liquid; A method for obtaining a concentrated solution having an increased concentration of extracellular vesicles by alternating tangential flow filtration, in which the first filtration step and the second filtration step are alternately performed multiple times, A method for separating and purifying extracellular vesicles, wherein the membrane surface velocity in the first filtration step and the second filtration step is 0.3 m / sec to 2 m / sec.

2. The method for separating and purifying extracellular vesicles according to claim 1, wherein the filtration method comprises filtering the mesenchymal stem cell culture supernatant through a microfiltration membrane having a pore size of 0.1 μm to 0.5 μm, and then using the filtrate from the microfiltration membrane, alternately performing the first filtration step and the second filtration step multiple times.

3. The method for separating and purifying extracellular vesicles according to claim 1 or 2, wherein when the first filtration step is carried out, a buffer solution is added to the mesenchymal stem cell culture supernatant or the second concentrate obtained in the second filtration step to dilute it, and then the first filtration step is carried out.

4. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 3, wherein the first filtration step and the second filtration step are carried out by pressurizing the filtration with a gas selected from nitrogen gas, an inert gas, carbon dioxide, and air filtered through a HEPA filter.

5. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 4, wherein the amount of protein contained in a culture supernatant of mesenchymal stem cells containing extracellular vesicles as a starting material is reduced to 1 / 5 or less.

6. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 5, wherein the insulin concentration in the separated and purified concentrate is 5 mg / L or less.

7. The amount of extracellular vesicles contained in the culture supernatant of the mesenchymal stem cells is measured using the ExoScreen method for the extracellular vesicles contained in the concentrate. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 6, wherein the amount of extracellular vesicles is concentrated by a factor of 5 or more and has a recovery rate of 50% or more.

8. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 7, 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.

9. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 8, wherein when the first filtration step and the second filtration step are performed alternately, the dilution factor of the object to be filtered in the first filtration step is increased as the number of times the steps are performed increases.

10. The method for separating and purifying extracellular vesicles according to any one of claims 1 to 8, wherein when the first filtration step and the second filtration step are alternately performed, the dilution factor of the object to be filtered in the first filtration step is increased in the range of 2 to 15 volume times as the number of times the steps are performed increases.

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