Method for producing extracellular vesicles

Abstract

This invention provides a method for producing extracellular vesicles with a high miRNA content. [Solution] A method for producing extracellular vesicles, comprising a culture step of culturing cells in a culture medium to obtain a culture supernatant containing extracellular vesicles, wherein the culture medium is FreeStyle(trademark) 293 expression medium.

Description

[Technical Field]

[0001] This disclosure relates to a method for producing extracellular vesicles. [Background technology]

[0002] There is a need for technologies to produce and detect extracellular vesicles (hereinafter referred to as "extracellular vesicles") that have a heterogeneous lipid bilayer structure released by cells. For example, Patent Document 1 discloses a method for detecting extracellular vesicles in a sample, which includes the steps of forming a complex between a carrier-bound T cell immunoglobulin / mucin domain-containing molecule 4 (Tim4) protein and extracellular vesicles in a sample in the presence of calcium ions, and detecting the complex using an antibody that binds to the extracellular vesicles. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2021-012200 [Overview of the project] [Problems that the invention aims to solve]

[0004] During the production of extracellular vesicles, cell culture is performed in basal media such as DMEM. However, there was a problem in that the production of extracellular vesicles containing microRNA was significantly reduced when using basal media (DMEM).

[0005] One aspect of this disclosure aims to provide a method for producing extracellular vesicles with a high microRNA content. [Means for solving the problem]

[0006] This disclosure provides the following aspects: [1] A method for producing extracellular vesicles, comprising a culture step of culturing cells in a culture medium to obtain a culture supernatant containing extracellular vesicles, wherein the culture medium is FreeStyle® 293 expression medium. [2] A method for producing extracellular vesicles according to [1], further comprising the steps of: contacting the culture supernatant with a solid support having a metal oxide on its surface in the presence of carbonate ions, phosphate ions, and calcium ions to adsorb the extracellular vesicles onto the solid support; separating the solid support from the liquid containing the culture supernatant; and desorbing the extracellular vesicles from the solid support. [3] The method for producing extracellular vesicles according to [1], further comprising the step of mixing a polymer flocculant with the culture supernatant to precipitate the extracellular vesicles. [4] A method for producing extracellular vesicles according to [1], further comprising the steps of: contacting the culture supernatant with a solid support containing phosphorus and calcium to adsorb the extracellular vesicles onto the solid support; separating the solid support from the liquid containing the culture supernatant; and desorbing the extracellular vesicles from the solid support. [5] The method according to any one of [1] to [4], further comprising a concentration step of concentrating the culture supernatant by ultrafiltration. [6] The method according to [5], wherein the concentration step is a step of concentrating the components that have not passed through the ultrafiltration membrane using an ultrafiltration membrane with a fractional molecular weight cutoff of 50 to 200 kDa. [7] The method according to any one of [1] to [6], further comprising the step of measuring the amount of microRNA in the extracellular vesicles relative to the amount of protein in the fraction of the recovered extracellular vesicles. [8] The method according to any one of [1] to [7], further comprising the step of measuring the amount of microRNA in the extracellular vesicles relative to the amount of extracellular vesicle markers, using the recovered extracellular vesicles as a measurement sample. [9] The method according to any one of [1] to [8], wherein the cells are human embryonic kidney cells. [Effects of the Invention]

[0007] According to one aspect of the present disclosure, a method for producing extracellular vesicles rich in microRNA can be provided.

Mode for Carrying Out the Invention

[0008] Hereinafter, exemplary embodiments of the present disclosure will be described. However, the present disclosure is not limited to the following embodiments. In this specification, a numerical range indicated by "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. Unless specifically stated, the units of the numerical values described before and after "~" are the same. Also, the individually described upper and lower limit values can be arbitrarily combined.

[0009] 〔Method for Producing Extracellular Vesicles〕 The method for producing extracellular vesicles according to this embodiment includes a culturing step of culturing cells in a medium to obtain a culture supernatant containing extracellular vesicles. In this culturing step, the medium is FreeStyle (trademark) 293 expression medium.

[0010] Extracellular vesicles are vesicles having a heterogeneous lipid bilayer structure released by cells. Extracellular vesicles can contain microRNA (microribonucleic acid; hereinafter sometimes referred to as "miRNA") that functions as an early diagnosis marker for cancer and the like, and CD63 (protein), etc.

[0011] <Culturing Step> In the culturing step, cells are cultured in a medium to obtain a culture supernatant containing extracellular vesicles. The medium in the culturing step is FreeStyle (trademark) 293 expression medium. FreeStyle (trademark) 293 expression medium is a medium provided by Thermo Fisher Scientific and is also used as a suspension culture medium for HEK293 cells. By using FreeStyle (trademark) 293 expression medium as the medium, the content of microRNA in the extracellular vesicles obtained by the method according to this embodiment increases.

[0012] Examples of cells include various cell lines such as CHO cells, L929 cells, human fetal kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells; epithelial cells and endothelial cells, which constitute various tissues and organs in the body; skeletal muscle cells, smooth muscle cells, and cardiomyocytes, which are contractile cells; neurons, glial cells, and fibroblasts, which constitute the nervous system; hepatocytes, non-parenchymal hepatocytes, and adipocytes, which are involved in the metabolism of the body; cells with differentiation potential and cells differentiated from cells with differentiation potential. Examples of cells with differentiation potential include stem cells present in various tissues, such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as pluripotent stem cells (pluripotent stem cells) such as ES cells and iPS cells.

[0013] The cells may be human embryonic kidney cells. Examples of human embryonic kidney cells include HEK293T, HEK293S, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, and HEK293MSR. HEK293T cells are preferred because they can increase the production of extracellular vesicles.

[0014] Culture conditions such as culture temperature, culture time, cell concentration, and culture vessel can be set to optimal conditions depending on the cell type, etc.

[0015] The culture temperature is not particularly limited as long as it is within the range at which the cells can grow, and may be, for example, 20-40°C, 25-40°C, 30-40°C, 35-40°C, or 36-38°C.

[0016] The incubation time may be 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours or more. The incubation time may be, for example, 216 hours or less, 168 hours or less, 144 hours or less, 120 hours or less, 96 hours or less, 72 hours or less, 48 ​​hours or less, or 24 hours or less.

[0017] The cell concentration in the culture medium is, for example, 1000 cells / cm² relative to the area of ​​the cell adhesion region of the container used for culture (for example, the bottom area of ​​a petri dish). 2 More than 5000 pieces / cm 2 More than 10000 pieces / cm 2 More than 100000 pieces / cm 2 More than or equal to 500,000 pieces / cm² 2 The above is sufficient, and 1,000,000 pieces / cm² 2 Below 100000 pieces / cm 2 The following, or 10,000 pieces / cm 2 The following is acceptable:

[0018] Culture vessels are not particularly limited, but examples include flasks, petri dishes, plates, cell culture bags, and large-scale culture devices.

[0019] If necessary, a pre-culture step may be performed before the culture step, in which cells are pre-cultured in a culture medium.

[0020] The culture medium for preliminary culture may be a basal medium. Examples of culture media for preliminary culture include DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, D-MEM / Ham's F12, MEM (Minimal Essential Medium), and RPMI-1640 medium. The culture medium for preliminary culture may also contain serum. Examples of serum include fetal bovine serum (FBS), horse serum, and calf serum.

[0021] The culture conditions in the preliminary culture step (culture temperature, culture time, culture vessel, and cell concentration in the culture medium) may be those described as conditions in the culture step. The preliminary culture temperature may be, for example, 35-40°C or 36-38°C. The preliminary culture time may be 24 hours or more, or 36 hours or more, and 72 hours or less, or 60 hours or less.

[0022] The method according to this embodiment may include centrifugation of the culture after the cultivation step. This allows for the removal of coarse aggregates from the culture after the cultivation step. Centrifugation may be performed multiple times under conditions of different centrifugal forces.

[0023] The centrifugal force in centrifugation may be, for example, 10 × g or more, 100 × g or more, or 300 × g or more, and may be 5000 × g or less, 1000 × g or less, or 500 × g or less. The centrifugation time may be 1 minute or more, 3 minutes or more, or 5 minutes or more, and may be 30 minutes or less, 20 minutes or less, or 10 minutes or less. Centrifugation may be performed under conditions such as 300 × g for 5 minutes, 1200 × g for 20 minutes, or 10000 × g for 30 minutes. The temperature of the culture supernatant during centrifugation may be 40°C or less, 37°C or less, or 30°C or less, and may be 4°C or higher, 10°C or higher, or 20°C or higher, for example, greater than 0°C and 5°C or 4°C.

[0024] The culture supernatant (such as the culture supernatant from which coarse aggregates have been removed by centrifugation) may be filtered. Filtering is beneficial because it more efficiently removes impurities and further improves the recovery rate of extracellular vesicles.

[0025] The pore size of the filter may be 0.1 μm or larger, or 0.5 μm or larger, or 5 μm or smaller, or 1 μm or smaller, or 0.22 μm.

[0026] <Concentration process> The method according to this embodiment may further include a concentration step of concentrating the culture supernatant. By concentrating the culture supernatant containing extracellular vesicles before the recovery step, the recovery rate of extracellular vesicles in the recovery step can be further increased.

[0027] The culture supernatant can be concentrated by ultrafiltration, ultracentrifugation, and / or drying. Ultrafiltration methods include membrane filtration, cross-flow filtration, batch filtration, continuous filtration, and centrifugal filtration. Membrane filtration is preferred for ultrafiltration. Ultracentrifugation conditions include, for example, 100,000 × g for 70 minutes. Drying can be done by natural drying or freeze-drying.

[0028] The concentration step is preferably a step in which components that have not passed through the ultrafiltration membrane are concentrated.

[0029] The molecular weight cutoff of the ultrafiltration membrane may be 50 kDa or higher, 70 kDa or higher, 90 kDa or higher, 110 kDa or higher, 130 kDa or higher, 150 kDa or higher, 170 kDa or higher, or 190 kDa or higher. A molecular weight cutoff of 90 kDa or higher is preferable because it allows for a greater reduction in the amount of impurities.

[0030] The molecular weight cutoff of the ultrafiltration membrane may be 200 kDa or less, 180 kDa or less, 160 kDa or less, 140 kDa or less, 120 kDa or less, 100 kDa or less, 80 kDa or less, or 60 kDa or less. A molecular weight cutoff of 120 kDa or less is preferable because it further increases the recovery rate of extracellular vesicles.

[0031] The concentration step is preferably a step in which components that have not passed through the ultrafiltration membrane are concentrated using an ultrafiltration membrane with a molecular weight cutoff of 50 to 200 kDa or 90 to 120 kDa.

[0032] The concentration ratio of the culture supernatant in the concentration step may be 20 times or more, 25 times or more, or 30 times or more. The concentration ratio of the culture supernatant in the concentration step may be 50 times or less, 45 times or less, 40 times or less, or 35 times or less.

[0033] <Recovery Process> The method according to this embodiment may further include a recovery step of recovering extracellular vesicles from the culture supernatant.

[0034] Methods for recovering extracellular vesicles from the culture supernatant include methods for adsorbing the extracellular vesicles onto a solid support and methods for precipitating the extracellular vesicles with a polymer flocculant.

[0035] <Recovery of extracellular vesicles using solid support> The recovery process may include an adsorption step of contacting the culture supernatant with a solid support to adsorb the extracellular vesicles in the culture supernatant onto the solid support, a separation step of separating the solid support from the liquid containing the culture supernatant, and a desorption step of desorbing the extracellular vesicles from the solid support, in order to further increase the recovery rate of extracellular vesicles.

[0036] (Adsorption process) In the adsorption process, the culture supernatant is brought into contact with a solid support, and the extracellular vesicles in the culture supernatant are adsorbed onto the solid support.

[0037] The solid phase support may be a material comprising a core and a surface layer. The shape of the solid phase support can be appropriately selected depending on the application, including particles, pellets, powders, granules, fibers, membranes, substrates, tubes, bags, rings, plates, etc. The solid phase support may also be used packed into a column. It is preferable that the solid phase support is a magnetically responsive material. Magnetic responsiveness allows for separation of the solid phase support using a magnet, which facilitates the separation of the solid phase support from the liquid.

[0038] The surface layer of the solid support may contain, for example, metal oxides and proteins. It is preferable that the solid support has metal oxides on at least a portion of its surface, as this further improves the adsorption and recovery rates of extracellular vesicles.

[0039] Examples of metal oxides include silicon dioxide (silica), glass, zinc oxide, titanium oxide, nickel oxide, aluminum oxide, yttria oxide, tin oxide, indium oxide and indium tin oxide, as well as ceramics such as zirconia and hydroxyapatite.

[0040] The metal oxide is preferably silicon dioxide (silica) because it more readily adsorbs extracellular vesicles. When the metal oxide is silicon dioxide (silica), it becomes easier to desorb extracellular vesicles at a pH near neutral, where denaturation of extracellular vesicles is less likely to occur, it becomes easier to adsorb extracellular vesicles in blood or samples containing serum or plasma, its acid resistance is further enhanced, and it becomes easier to recover extracellular vesicles with a high microRNA content.

[0041] The surface layer may further contain minute magnetic materials. The minute magnetic materials are magnetically responsive and smaller than the core particles. The particle size of the minute magnetic materials may be 1 μm or less, 0.80 μm or less, 0.60 μm or less, 0.40 μm or less, 0.30 μm or less, 0.20 μm or less, or 0.15 μm or less, and may be 0.001 μm or more, 0.005 μm or more, or 0.008 μm or more. Since it is preferable that the material has superparamagnetism, the particle size of the minute magnetic materials is preferably 0.001 to 1 μm, more preferably 0.001 to 0.5 μm, even more preferably 0.001 to 0.1 μm, and particularly preferably 0.001 to 0.05 μm. The minute magnetic materials are preferably nano-sized magnetic materials (nanomagnetic materials).

[0042] The particle size of a minute magnetic material can be calculated by measuring the maximum diameter of 10 or more particles in an image observed with a transmission electron microscope and then calculating the average value.

[0043] Examples of materials for minute magnetic materials include iron oxides such as magnetite.

[0044] The micromagnetic material may contain a cationic dispersant or an anionic dispersant on its surface.

[0045] Commercially available micromagnetic materials can be used. An example of a commercially available micromagnetic material is EMG607 (trade name, manufactured by Ferrotec).

[0046] The minute magnetic material may be dispersed in the surface layer, or it may be in a state where it covers the core and the layer made of metal oxide between the core and the layer made of metal oxide. The acid resistance of the solid support can be improved by having the minute magnetic material dispersed in the surface layer or by having the surface of the minute magnetic material covered with metal oxide.

[0047] Since the magnetic responsiveness is further improved, the content of fine magnetic material per gram of solid support may be 0.1 g / g or more, 0.2 g / g or more, 0.5 g / g or more, or 0.7 g / g or more. Since the dispersibility of the solid support in aqueous solvents is further improved, the content of fine magnetic material per gram of solid support may be 0.9 g / g or less, 0.8 g / g or less, 0.7 g / g or less, or 0.6 g / g or less.

[0048] From the viewpoint of controlling the specific gravity of the solid phase support and facilitating adjustment of the settling rate of the solid phase support, the weight percentage of minute magnetic material contained in the solid phase support may be 5-70% by mass, 10-60% by mass, 15-50% by mass, or 20-40% by mass, based on the total mass of the solid phase support. The content of minute magnetic material can be measured by ICP (Inductively Coupled Plasma) emission spectrometry.

[0049] The thickness of the surface layer may be 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, or 0.02 μm or more, from the viewpoint of further improving acid resistance. From the viewpoint of increasing the specific surface area of ​​the solid phase support, the thickness of the surface layer may be 0.1 μm or less, or 0.05 μm or less. For example, the thickness of the surface layer may be 0.001 μm or more and 0.1 μm or less. The thickness of the surface layer can be calculated by measuring the thickness of the surface layer for 10 or more solid phase supports in an image of the cross-section of the solid phase support observed with a transmission electron microscope, and calculating the average value.

[0050] The core shape can be appropriately selected from parts, pellets, powders, granules, fibers, membranes, substrates, tubes, bags, rings, plates, etc.

[0051] Examples of particulate core shapes include nearly spherical shapes, non-spherical shapes such as ellipsoids, cubes, cylinders, and polygonal prisms, and porous particles with large surface area and irregularities such as those with protrusions. Since magnetic particles with good magnetic responsiveness are easily obtained, the core particle size may be 0.1 μm or larger, 0.5 μm or larger, 1.0 μm or larger, 1.5 μm or larger, or 2.0 μm or larger. Since magnetic particles with good redispersibility in aqueous solvents are easily obtained, the core particle size may be 100 μm or less, 80 μm or less, 60 μm or less, 40 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, or 3 μm or less. The core particle size may be 0.1 μm or larger and 100 μm or smaller, 0.5 μm or larger and 10 μm or smaller, 1.0 μm or larger and 5.0 μm or smaller, or 2.0 μm or larger and 3.0 μm or smaller. The particle size of the core can be calculated by determining the mode diameter from the particle size distribution measured by dynamic light scattering.

[0052] The core may be composed of a polymer or a metal oxide. When the core is composed of a polymer, the specific gravity of the solid support can be reduced compared to when the main component of the core is a component with a high specific gravity (magnetic material or metal oxide), thereby improving the dispersibility of the solid support in the solvent. This improved dispersibility makes it easier to obtain a particulate solid support that can be uniformly dispersed with less stirring force. A particulate solid support that can be uniformly dispersed can improve the reproducibility of extracellular vesicle purification.

[0053] The metal oxide used as a component of the core may be any of the materials exemplified as metal oxides in the surface layer.

[0054] The polymer may contain, as monomer units, at least one selected from the group consisting of, for example, styrene monomer units, (meth)acrylate monomer units, and vinyl ester monomer units.

[0055] Examples of styrene monomers include styrene, α-methylstyrene, vinyltoluene, p-methylstyrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, 4-tert-butylstyrene, 3,4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4-chloro-3-methylstyrene, divinylbenzene, and sodium p-styrenesulfonate.

[0056] Examples of (meth)acrylate monomers include (meth)acrylate ((meth)acrylic acid); alkyl (meth)acrylates such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, and 2-ethylhexyl (meth)acrylate; (cyclo)alkyl (meth)acrylates such as cyclohexyl (meth)acrylate; 2-methoxyethyl (meth)acrylate, p-methoxycyclohexyl (meth)acrylate ) Acrylates and other alkoxy(cyclo)alkyl(meth)acrylates; polyvalent(meth)acrylates such as trimethylolpropane(meth)acrylate; cyanoacrylates such as 2-cyanoethyl(meth)acrylate, 2-cyanopropyl(meth)acrylate, 3-cyanopropyl(meth)acrylate; hydroxymethyl(meth)acrylate, 2-hydroxyethyl(meth)acrylate, 6-hydroxyhexyl(meth)acrylate, 4-hydroxy(meth)acrylate Examples include substituted hydroxy(meth)acrylates such as cyclohexyl(meth)acrylate, neopentyl glycol mono(meth)acrylate, 3-chloro-2-hydroxypropyl(meth)acrylate, and 3-amino-2-hydroxypropyl(meth)acrylate; glycidyl group-containing acrylates such as glycidyl(meth)acrylate, methylglycidylmethyl acrylate, and epoxidized cyclohexyl(meth)acrylate; and polyfunctional(meth)acrylates such as trimethylolpropaneethoxytriacrylate, pentaerythritolethoxytetraacrylate, trimethylolpropanepropoxytriacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, dipentaerythritol hexaacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, tricyclodecanedimethanol diacrylate, and ethoxylated phenyl acrylate. (Meth)acrylates may be crosslinked.

[0057] Examples of vinyl ester monomers include vinyl acetate, vinyl propionate, and vinyl versatate.

[0058] A solid-state support comprising a core and a surface layer can be obtained, for example, by a method including forming a surface layer on at least a portion of the surface of the core. A solid-state support in which the surface layer comprises a metal oxide and a micromagnetic material can be obtained, for example, by a method including a magnetization step of adsorbing (physical adsorption) the micromagnetic material onto the surface of the core, and a coating step of coating the core with the adsorbed micromagnetic material with a metal oxide.

[0059] The magnetization process can be carried out, for example, by a method that includes reacting the core and minute magnetic material in an aqueous solution to adsorb the minute magnetic material onto the core, drying the core with the adsorbed minute magnetic material, and then compounding the core and minute magnetic material by applying mechanical energy.

[0060] In the magnetization process, the amount of minute magnetic material used may be, for example, 0.5g or more, 1.0g or more, or 1.1g or more, or 2.0g or less, or 1.5g or less, per 1g of core. The drying conditions in the magnetization process can be set appropriately according to the type of material used, the composite conditions, etc. The drying temperature may be, for example, 40°C or more, or 45°C or more, or 80°C or less, or 65°C or less. The drying time may be, for example, 1 hour or more, 5 hours or more, or 10 hours or more, or 25 hours or less, or 20 hours or less.

[0061] As a method for compounding the core and minute magnetic material by applying mechanical energy, a method that allows for dry processing of the material is preferred, such as using a device that performs compounding by swirling force in a high-speed airflow, a device that performs mixing and stirring using a rotation-orbit mixer or ball mill, or a device that performs surface coating using a spray dryer. Examples of devices for compounding by applying mechanical energy include the hybridization system NHS series (manufactured by Nara Machine Works Co., Ltd.), Nobilta NOB (manufactured by Hosokawa Micron Corporation), high-speed stirring type powder spheroidization device NSM series (manufactured by Seishin Corporation), and mini spray dryer B-290 type (Shibata Scientific Co., Ltd.).

[0062] When performing compounding by the swirling force in a high-speed air flow, since it is suitable for firmly fixing the micro-magnetic bodies to the core part, the rotation speed is 8000 min -1 or more, 9000 min -1 or more, 10000 min -1 or more, or 11000 min -1 or more. Since it is suitable for shaping the particle shape into a spherical shape, the rotation speed is 15000 min -1 or less, 14000 min -1 or less, 13000 min -1 or less, or 12000 min -1 or less. Since it is suitable for shaping the particle shape into a spherical shape, the treatment time may be 1 to 60 minutes, 1 to 20 minutes, 1 to 10 minutes, or 3 to 10 minutes. Since it is suitable for suppressing the fusion of the particles, the treatment temperature may be 25 to 80 °C, 25 to 60 °C, or 25 to 50 °C, or 25 to 40 °C.

[0063] As a method of coating the core part with a metal oxide in the coating process, there is no particular limitation, but a method of forming a metal oxide on the surface of the core part by dispersing the core part adsorbed with the micro-magnetic bodies in a solution having a reagent as a raw material of the metal oxide and a reaction catalyst and allowing the reaction to proceed, a method of forming a metal oxide on the surface of the core part by dispersing the core part adsorbed with the micro-magnetic bodies and the powder of the metal oxide in a high-speed air flow, a method of coating the surface of the core part with a metal oxide or its raw material, etc. can be mentioned.

[0064] As a method for coating the core with a metal oxide, a sol-gel method is preferably used, in which the core with adsorbed micromagnetic material is dispersed in a solvent, and then a metal oxide precursor and a reaction catalyst are added. Examples of metal oxide precursors include metal alkoxides. As precursors to metal oxides, for example, in the case of silica, tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate can be used; in the case of zinc oxide, zinc methoxide, zinc ethoxide, zinc propoxide, and zinc butoxide can be used; in the case of titanium oxide, tetramethyl orthotitanate, tetraethyl orthotitanate, tetrapropyl orthotitanate, and tetrabutyl orthotitanate can be used; in the case of yttria oxide, yttrium methoxide, yttrium ethoxide, yttrium propoxide, and yttrium butoxide can be used; in the case of tin oxide, tin methoxide, tin ethoxide, tin propoxide, and tin butoxide can be used; and in the case of indium oxide, indium methoxide, indium ethoxide, indium propoxide, and indium butoxide can be used. In the case of indium tin oxide, a mixture of the tin oxide precursor and the indium oxide precursor can be used. As a reaction catalyst, water or aqueous ammonia can be used. The amount of metal oxide precursor added during the sol-gel reaction may be 0.05 g or more, 0.1 g or more, 0.2 g or more, or 0.5 g or more per 1 g of core, as this is suitable for forming a sufficient amount of metal oxide on the surface of the core. The amount of metal oxide precursor added during the sol-gel reaction may be 5 g or less, 2 g or less, 1 g or less, or 0.5 g or less per 1 g of core, as this is suitable for suppressing aggregation of the core during the reaction. The amount of reaction catalyst added during the reaction may be 20 mL or more, 30 mL or more, 40 mL or more, or 50 mL or more per 1 L of solvent, as this is suitable for forming a sufficient amount of metal oxide on the surface of the core. The amount of reaction catalyst added during the reaction may be 100 mL or less, 80 mL or less, 70 mL or less, or 60 mL or less per 1 L of solvent, as this is suitable for suppressing aggregation of the core during the reaction.

[0065] The reaction solvent used in forming the metal oxide may be ethanol, 2-propanol, or butanol, as these are suitable for uniformly coating the core with the metal oxide while suppressing aggregation.

[0066] The solid phase support may contain phosphorus and calcium elements, as this can further increase the recovery rate of extracellular vesicles. That is, the adsorption step may be a step of contacting the culture supernatant with the solid phase support containing phosphorus and calcium elements to adsorb the extracellular vesicles in the culture supernatant onto the solid phase support. When the solid phase support contains phosphorus and calcium elements, a fraction of extracellular vesicles is more likely to be obtained in which the amount of microRNA in the extracellular vesicles is greater relative to the amount of protein in the recovered extracellular vesicle fraction, and the amount of microRNA in the extracellular vesicles is greater relative to the amount of extracellular vesicle markers.

[0067] A solid-phase support containing phosphorus and calcium is a compound containing phosphorus and calcium as constituent elements, or a mixture thereof. The solid-phase support may further contain at least one element selected from the group consisting of oxygen and calcium.

[0068] The solid support containing phosphorus and calcium elements may be a reaction product of raw materials containing at least phosphate and calcium salts. The raw materials for obtaining the reaction product may further contain carbonates. The forms of the phosphate, calcium salt and carbonate may be as described above.

[0069] Solid phase supports containing phosphorus and calcium elements can be obtained by a reaction in a reaction solution containing a raw material containing phosphates and calcium salts, and optionally carbonates, and water. Examples of water used herein include distilled water, pure water, and ultrapure water (such as Milli-Q water). Milli-Q water refers to ultrapure water obtained using a Milli-Q water production system from Merck Millipore (Merck GmbH).

[0070] The concentration of phosphate in the reaction solution may be 90 mg / L or higher, 100 mg / L or higher, 200 mg / L or higher, 400 mg / L or higher, 600 mg / L or higher, 800 mg / L or higher, or 1000 mg / L or higher, based on the total volume of the reaction solution, in order to obtain a solid support with superior recovery of extracellular vesicles. The concentration of phosphate in the reaction solution may be 10000 mg / L or lower, 8000 mg / L or lower, 6000 mg / L or lower, 4000 mg / L or lower, 3000 mg / L or lower, 2000 mg / L or lower, 1500 mg / L or lower, 1300 mg / L or lower, or 1100 mg / L or lower, based on the total volume of the reaction solution, in order to obtain a solid support with superior recovery of extracellular vesicles. The concentration of phosphate in the reaction solution may be 90-10000 mg / L, 200-4000 mg / L, 500-3000 mg / L, or 800-1500 mg / L based on the total volume of the reaction solution, as this makes it easier to obtain a solid support with superior extracellular vesicle recovery.

[0071] The concentration of calcium salt in the reaction solution may be 200 mg / L or more, 400 mg / L or more, 600 mg / L or more, 800 mg / L or more, 1000 mg / L or more, 1200 mg / L or more, 1400 mg / L or more, 1600 mg / L or more, 1800 mg / L or more, or 1900 mg / L or more, based on the total volume of the reaction solution, as this makes it easier to obtain a solid support with superior recovery of extracellular vesicles. The concentration of calcium salt in the reaction solution may be 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, 4,000 mg / L or less, 3,000 mg / L or less, 2,500 mg / L or less, 2,400 mg / L or less, 2,300 mg / L or less, 2,200 mg / L or less, or 2,100 mg / L or less, based on the total volume of the reaction solution, in order to obtain a solid support with superior recovery of extracellular vesicles. The concentration of calcium salt in the reaction solution may be 200 to 10,000 mg / L, 600 to 6,000 mg / L, 1,000 to 4,000 mg / L, 1,400 to 3,000 mg / L, or 1,800 to 2,200 mg / L, based on the total volume of the reaction solution, in order to obtain a solid support with superior recovery of extracellular vesicles.

[0072] The carbonate concentration in the reaction solution may be 1000 mg / L or higher, 5000 mg / L or higher, 10000 mg / L or higher, 15000 mg / L or higher, 20000 mg / L or higher, 25000 mg / L or higher, 30000 mg / L or higher, 32000 mg / L or higher, 34000 mg / L or higher, or 36000 mg / L or higher, based on the total volume of the reaction solution, as this makes it easier to obtain a solid support with superior recovery of extracellular vesicles. The carbonate concentration in the reaction solution may be 100000 mg / L or lower, 60000 mg / L or lower, 50000 mg / L or lower, 45000 mg / L or lower, 40000 mg / L or lower, or 38000 mg / L or lower, based on the total volume of the reaction solution, as this makes it easier to obtain a solid support with superior recovery of extracellular vesicles. The concentration of carbonate in the reaction solution may be 1,000-100,000 mg / L, 5,000-60,000 mg / L, 10,000-50,000 mg / L, 2,000-45,000 mg / L, 30,000-40,000 mg / L, or 34,000-38,000 mg / L based on the total volume of the reaction solution, as this makes it easier to obtain a solid support with superior recovery of extracellular vesicles.

[0073] The temperature of the reaction solution during the reaction may be 20-30°C (room temperature). The reaction time may be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, and 36 hours or less, 30 hours or less, 24 hours or less, 18 hours or less, 12 hours or less, 6 hours or less, or 3 hours or less. For example, it may be 1-24 hours or 1-4 hours.

[0074] The liquid containing the solid support obtained after the reaction may be used directly for the purification of extracellular vesicles, or at least a portion of the liquid component may be removed before use. As a method for removing at least a portion of the liquid component, known solid-liquid separation methods including centrifugation and subsequent removal of the supernatant can be used.

[0075] The solid phase support may be mixed with the culture supernatant in a solid state, or an aqueous solution containing the solid phase support may be prepared in advance and mixed with the culture supernatant in that aqueous solution state.

[0076] Contact between the culture supernatant and the solid support can be achieved, for example, by adding the solid support to the culture supernatant and then stirring to disperse the solid support in the liquid. The adsorption time is not particularly limited, but to allow the extracellular vesicles in the culture supernatant to come into sufficient contact with the surface of the solid support, it may be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, or 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more. To facilitate the suppression of extracellular vesicle degeneration, the adsorption time may be 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or 30 minutes or less, 20 minutes or less, or 10 minutes or less.

[0077] In the adsorption process, to further increase the adsorption rate of extracellular vesicles, the amount of solid support added may be 1 mg or more, 2 mg or more, 5 mg or more, or 8 mg or more per 1 mL of culture supernatant. To suppress contamination by impurities derived from the solid support, the amount of solid support added may be 100 mg or less, 75 mg or less, 50 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, or 12 mg or less per 1 mL of culture supernatant.

[0078] When the solid support is a solid support having a metal oxide on its surface, the adsorption step is preferably carried out in the presence of carbonate ions, phosphate ions, and calcium ions, as this further improves the adsorption rate and recovery rate of extracellular vesicles. That is, the adsorption step may be a step in which the culture supernatant and the solid support having a metal oxide on its surface are brought into contact in the presence of carbonate ions, phosphate ions, and calcium ions, and extracellular vesicles are adsorbed onto the solid support.

[0079] The carbonate ion content (moles) per 1 mol of phosphate ions may be 0.1 mol or more, 0.5 mol or more, 1 mol or more, 2 mol or more, 5 mol or more, 10 mol or more, 15 mol or more, 20 mol or more, 25 mol or more, 30 mol or more, 35 mol or more, 40 mol or more, 45 mol or more, 50 mol or more, or 55 mol or more, in order to further promote the adsorption of extracellular vesicles to the solid support and to further improve the recovery rate of extracellular vesicles. The carbonate ion content (moles) per 1 mol of phosphate ions may be 200 mol or less, 150 mol or less, 120 mol or less, 80 mol or less, or 60 mol or less, in order to further promote the adsorption of extracellular vesicles to the solid support and to further improve the recovery rate of extracellular vesicles.

[0080] The carbonate ion content (moles) per 1 mol of calcium ions may be 0.05 mol or more, 0.15 mol or more, 0.3 mol or more, 1 mol or more, 3 mol or more, 6 mol or more, 8 mol or more, 10 mol or more, 12 mol or more, 15 mol or more, 18 mol or more, 20 mol or more, or 22 mol or more, in order to further promote the adsorption of extracellular vesicles and to further improve the recovery rate of extracellular vesicles. The carbonate ion content (moles) per 1 mol of calcium ions may be 150 mol or less, 120 mol or less, 80 mol or less, 70 mol or less, 60 mol or less, 55 mol or less, 50 mol or less, 45 mol or less, 40 mol or less, 35 mol or less, 30 mol or less, or 25 mol or less, in order to further promote the adsorption of extracellular vesicles and to further improve the recovery rate of extracellular vesicles.

[0081] The amount of calcium ions (moles) per 1 mole of phosphate ions may be 0.1 mol or more, 0.3 mol or more, 0.5 mol or more, 0.8 mol or more, 1 mol or more, 1.2 mol or more, 1.4 mol or more, 1.5 mol or more, 1.6 mol or more, 1.8 mol or more, 2.0 mol or more, or 2.2 mol or more, in order to further promote the adsorption of extracellular vesicles and to further improve the recovery rate of extracellular vesicles. The amount of calcium ions (moles) per 1 mole of phosphate ions may be 20 mol or less, 18 mol or less, 16 mol or less, 14 mol or less, 12 mol or less, 10 mol or less, 8 mol or less, 6 mol or less, 4 mol or less, 3 mol or less, or 2.5 mol or less, in order to further promote the adsorption of extracellular vesicles and to further improve the recovery rate of extracellular vesicles.

[0082] The total number of moles of carbonate ions, phosphate ions, and calcium ions per 1 g of solid support may be 0.01 mmol or more, 0.05 mmol or more, 0.1 mmol or more, 0.5 mmol or more, 1 mmol or more, 2 mmol or more, 3 mmol or more, 4 mmol or more, 5 mmol or more, 10 mmol or more, 100 mmol or more, or 1000 mmol or more. The total number of moles of carbonate ions, phosphate ions, and calcium ions per 1 g of solid support may be 2000 mmol or less, 200 mmol or less, 20 mmol or less, 15 mmol or less, 8 mmol or less, 7 mmol or less, 6 mmol or less, 5 mmol or less, 2 mmol or less, 0.8 mmol or less, 0.5 mmol or less, 0.2 mmol or less, 0.08 mmol or less, or 0.05 mmol or less.

[0083] One method for performing the adsorption process in the presence of carbonate ions, phosphate ions, and calcium ions is to prepare a solid support dispersion containing carbonate ions, phosphate ions, calcium ions, and a solid support in advance, and then mix this aqueous solution with the culture supernatant.

[0084] A solid-phase support dispersion can be prepared, for example, by dissolving carbonates, phosphates, and calcium salts in a dispersion of a solid-phase support in a solvent.

[0085] The ratio of the amount of solid support dispersion used (mL) to the amount of culture supernatant used (mL) may be 0.5 or more, or 0.8 or more, and may be 2.0 or less, 1.5 or less, or 1.2 or less.

[0086] Carbonates are CO3 2- or HCO3 - It is a compound consisting of CO3 and its counterion. 2- or HCO3 - Examples of counterions include alkali metal ions (e.g., lithium ions, sodium ions, and potassium ions), alkaline earth metal ions (e.g., magnesium ions, calcium ions, strontium ions, and barium ions), and ammonium ions. Examples of carbonates include sodium bicarbonate, calcium carbonate, magnesium carbonate, potassium carbonate, lithium carbonate, ammonium carbonate, strontium carbonate, and barium carbonate. Sodium bicarbonate is preferred as the carbonate because it is suitable for increasing the adsorption rate of extracellular vesicles, suppressing losses during washing, and increasing the recovery rate of extracellular vesicles from the solid support.

[0087] Phosphates are PO4 3- HPO4 2- or H2PO 4- It is a compound consisting of a ion and its counterion. PO4 3- HPO4 2- or H2PO 4-Examples of counterions include alkali metal ions and alkaline earth metal ions. Examples of phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, calcium hydrogen phosphate, potassium hydrogen phosphate, magnesium hydrogen phosphate, ammonium hydrogen phosphate, strontium hydrogen phosphate, and barium hydrogen phosphate. The phosphate is preferably at least one selected from the group consisting of dihydrogen phosphates and hydrogen phosphates, or at least one selected from the group consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate, and more preferably sodium dihydrogen phosphate, as it is suitable for increasing the adsorption rate of extracellular vesicles, suppressing loss during washing, and increasing the recovery rate of extracellular vesicles from the solid support.

[0088] Calcium salts are Ca 2+ It is a compound consisting of Ca and its counterion. 2+ Examples of counterions include inorganic acid ions (e.g., halide ions, nitrate ions, sulfate ions) or organic acid ions (e.g., acetate ions). Examples of calcium salts include calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium sulfate, and calcium acetate. Calcium chloride is preferred as the calcium salt because it is suitable for increasing the adsorption rate of extracellular vesicles, suppressing losses during washing, and improving the recovery rate of extracellular vesicles from the solid support.

[0089] The total amount of carbonate, phosphate, and calcium salt added is preferably 1 mg / L or more, based on the total volume of the culture supernatant and the solid support dispersion, in order to promote the adsorption of extracellular vesicles. It may also be 10 mg / L or more, 100 mg / L or more, 200 mg / L or more, 500 mg / L or more, 1000 mg / L or more, 2000 mg / L or more, 3000 mg / L or more, 3500 mg / L or more, or 3800 mg / L or more. To facilitate the desorption of adsorbed extracellular vesicles, the concentrations of these at the time of adsorption may be 10000 mg / L or less, 8000 mg / L or less, 6000 mg / L or less, 5000 mg / L or less, or 4200 mg / L or less.

[0090] The carbonate concentration may be 50 mg / L or more, 80 mg / L or more, 100 mg / L or more, 500 mg / L or more, 1000 mg / L or more, 1500 mg / L or more, 2000 mg / L or more, 2500 mg / L or more, 3000 mg / L or more, or 3500 mg / L or more, based on the total volume of the culture supernatant and the solid support dispersion, and may be 500,000 mg / L or less, 400,000 mg / L or less, 300,000 mg / L or less, 200,000 mg / L or less, 100,000 mg / L or less, 50,000 mg / L or less, 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, or 4,000 mg / L or less.

[0091] The phosphate concentration may be 1 mg / L or more, 5 mg / L or more, 10 mg / L or more, 20 mg / L or more, 40 mg / L or more, 60 mg / L or more, 80 mg / L or more, 90 mg / L or more, or 100 mg / L or more, based on the total volume of the culture supernatant and the solid support dispersion, and may be 20,000 mg / L or less, 15,000 mg / L or less, 12,000 mg / L or less, 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, 4,000 mg / L or less, 2,000 mg / L or less, 1,500 mg / L or less, 1,200 mg / L or less, 1,000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, 200 mg / L or less, 150 mg / L or less, or 120 mg / L or less.

[0092] The calcium salt concentration may be 1 mg / L or more, 5 mg / L or more, 10 mg / L or more, 20 mg / L or more, 40 mg / L or more, 60 mg / L or more, 80 mg / L or more, 90 mg / L or more, 100 mg / L or more, 140 mg / L or more, 160 mg / L or more, or 180 mg / L or more, based on the total volume of the culture supernatant and the solid phase carrier dispersion, and may be 30,000 mg / L or less, 25,000 mg / L or less, or 20,000 mg / L or more. It may be less than or equal to g / L, less than or equal to 15,000 mg / L, less than or equal to 12,000 mg / L, less than or equal to 10,000 mg / L, less than or equal to 8,000 mg / L, less than or equal to 6,000 mg / L, less than or equal to 4,000 mg / L, less than or equal to 2,500 mg / L, less than or equal to 2,000 mg / L, less than or equal to 1,500 mg / L, less than or equal to 1,200 mg / L, less than or equal to 1,000 mg / L, less than or equal to 800 mg / L, less than or equal to 600 mg / L, less than or equal to 400 mg / L, less than or equal to 300 mg / L, or less than or equal to 250 mg / L.

[0093] (separation process) In the separation step, the solid support to which the extracellular vesicles are adsorbed is separated from the liquid. The separation method is not particularly limited, but separation by magnet is preferred due to its ease of operation. The separation method may also be a combination of separation by magnet and, if necessary, centrifugation, filtration, or natural sedimentation.

[0094] (Washing process) After the separation step, a washing step may be performed to wash the solid support to which the extracellular vesicles have been adsorbed. Examples of liquids for washing the solid support (washing solution) include buffer solutions, aqueous solutions containing carbonate ions, phosphate ions, and calcium ions, and acidic or alkaline liquids. Examples of buffer solutions include acetate buffer, phosphate buffer, citrate buffer, Tris-HCl buffer, and HEPES buffer. The pH of the buffer solution used as the washing solution may be 5.0 to 10.0, 6.0 to 10.0, greater than 7.0 and 10.0 or less, or 8.0 to 10.0 or less. The acidic or alkaline liquid may be a liquid within the above pH range.

[0095] The washing method is not particularly limited, but examples include mixing the solid support with the washing solution and stirring the washing solution. The temperature of the washing solution in the washing process may be 10-40°C or 15-25°C. The washing time (stirring time of the washing solution, etc.) in the washing process can be appropriately selected depending on the type of washing solution, the temperature of the washing solution, etc. The washing time may be, for example, 1 minute or more, 3 minutes or more, 30 minutes or less, 15 minutes or less, or 10 minutes or less.

[0096] The separation and washing steps can be repeated in this order to repeatedly wash the solid support to which the extracellular vesicles are adsorbed with the washing solution. By repeatedly performing the separation and washing steps, more residues other than the extracellular vesicles adsorbed on the solid support can be removed.

[0097] (Desorption process) In the desorption process, extracellular vesicles are desorbed from the solid support to which they are adsorbed. Methods for desorbing extracellular vesicles from a solid support include bringing the solid support to which the extracellular vesicles are adsorbed into contact with a liquid (desorption solution) for desorbing the extracellular vesicles.

[0098] The desorption solution is preferably a liquid containing a chelating agent. In this specification, "chelating agent" means a compound (polydentate ligand) that has multiple coordination sites within its molecule and coordinates polydentately to a metal ion, and "chelating action" means polydentate coordination (binding) to a metal ion.

[0099] The chelating agent may be at least one selected from the group consisting of polycarboxylic acid compounds, polyphosphate compounds, polyphenol compounds, other compounds having chelating properties, and salts thereof.

[0100] Polycarboxylic acid compounds are compounds having multiple carboxyl groups. A polycarboxylic acid compound may be at least one selected from the group consisting of aminopolycarboxylic acids having at least one amino group and multiple carboxyl groups, and polycarboxylic acids (non-aminopolycarboxylic acids) that do not contain an amino group but have multiple carboxyl groups. An aminopolycarboxylic acid may be at least one selected from the group consisting of EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), NTA (nitrilotriacetic acid), EGTA (ethylene glycol bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid), BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), and HEDTA (hydroxyethylethylenediaminetriacetic acid). A non-aminopolycarboxylic acid may be at least one selected from the group consisting of oxalic acid, polyacrylic acid, and citric acid.

[0101] Polyhydric phosphate compounds are compounds having multiple phosphate groups. Polyhydric phosphate compounds may be phytic acid. Polyhydric phenolic compounds are compounds having multiple phenolic hydroxyl groups. Polyhydric phenolic compounds may be at least one selected from the group consisting of polyphenols and calixarenes. Other chelating compounds may be at least one selected from the group consisting of desferrioxamine and penicillamine.

[0102] Since it is more suitable for promoting the desorption of extracellular vesicles from the solid support, the chelating agent may be a polycarboxylic acid compound or an aminopolycarboxylic acid.

[0103] The chelating agent content in the desorption solution may be 0.01 mmol / L or more, 0.1 mmol / L or more, 0.3 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 2 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 50 mmol / L or more, or 100 mmol / L or more, based on the total volume of the desorption solution, in order to promote the desorption of extracellular vesicles. The chelating agent content in the desorption solution may be 1000 mmol / L or less, 500 mmol / L or less, 300 mmol / L or less, 150 mmol / L or less, 80 mmol / L or less, 60 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 15 mmol / L or less, 8 mmol / L or less, 6 mmol / L or less, 30 mmol / L or less, 15 mmol / L or less, 8 mmol / L or less, 6 mmol / L or less, 3 mmol / L or less, 1.5 mmol / L or less, 0.8 mmol / L or less, 0.4 mmol / L or less, 0.2 mmol / L or less, or 0.05 mmol / L or less, based on the total volume of the desorption solution, in order to promote the desorption of extracellular vesicles and suppress the degeneration of extracellular vesicles.

[0104] The pH of the desorption solution may be 6 or higher, and may be 7 or higher, 8 or higher, 9 or higher, 10 or higher, 11 or higher, 12 or higher, 13 or higher, or 14 or higher in order to further increase the desorption rate of extracellular vesicles. The pH of the liquid used in the desorption process may be 14 or lower, 13 or lower, 12 or lower, 11 or lower, 10 or lower, 9 or lower, or 8 or lower in order to suppress the denaturation of extracellular vesicles.

[0105] The desorption solution may be an alkaline liquid. In this case, extracellular vesicles are more easily desorbed from the solid support.

[0106] There are no particular limitations on the method for adjusting the pH of the desorption solution, but one method is to add a base such as sodium hydroxide, sodium phosphate, sodium citrate, sodium carbonate, or sodium bicarbonate.

[0107] The desorption time is not particularly limited, but may be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, or 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more, in order to sufficiently desorb the extracellular vesicles in the liquid from the surface of the solid support. The desorption time may be 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or 30 minutes or less, 20 minutes or less, or 10 minutes or less, in order to suppress the degeneration of the extracellular vesicles.

[0108] <Recovery of extracellular vesicles using polymer flocculants> The recovery step may include a precipitation step in which the extracellular vesicles are reacted with the polymeric flocculant in a mixture containing the culture supernatant and the polymeric flocculant to allow the extracellular vesicles to settle, as this can further increase the recovery rate of extracellular vesicles.

[0109] Examples of polymer flocculants include cationic polymer flocculants, anionic polymer flocculants, and nonionic polymer flocculants.

[0110] The polymer flocculant may include polymers such as polyethylene glycol, polyacrylamide, polystyrene sulfonic acid, polyethyleneimine, casein, and chitosan. In this specification, "polyethylene glycol" refers to a polymer with the formula: H-[O-CH2-CH2] n It is a polymer represented by -OH, and includes polyethylene glycol (PEG), which has a molecular weight of less than 20,000, and polyethylene oxide (PEO), which has a molecular weight of 20,000 or more.

[0111] The polymer flocculant is preferably polyethylene glycol because it can further increase the recovery rate of extracellular vesicles.

[0112] The number-average molecular weight of the polymer flocculant (e.g., polyethylene glycol) may be 1000 or more, 2000 or more, 3000 or more, 5000 or more, 10000 or more, 20000 or more, 30000 or more, or 40000 or more. The above number-average molecular weight is preferably 3000 or more because it can further increase the recovery rate of extracellular vesicles. The above number-average molecular weight may be 50000 or less, 35000 or less, 25000 or less, 15000 or less, 8000 or less, 6000 or less, 4000 or less, 2500 or less, or 1500 or less. The above number-average molecular weight is preferably 10000 or less because it can increase the recovery rate of extracellular vesicles.

[0113] The above number-average molecular weight is measured by gel permeation chromatography (GPC) and is converted to standard polyethylene glycol / oxide (PEG / PEO).

[0114] The amount of polymer flocculant added per 1 mL of culture supernatant may be 0.1 mL or more, 0.3 mL or more, 0.5 mL or more, or 1 mL or more, and may be 2 mL or less, 1.5 mL or less, or 0.7 mL or less.

[0115] The conditions for contacting the culture supernatant with the polymer flocculant can be appropriately selected depending on the type of polymer flocculant. For example, the temperature of the mixture containing the culture supernatant and the polymer flocculant may be 1°C or higher, 3°C or higher, or 10°C or higher, and may be 20°C or lower, 15°C or lower, or 5°C or lower. The time for maintaining the temperature of the mixture within the above numerical range may be 1 hour or more, 5 hours or more, or 10 hours or more, and may be 24 hours or less, 12 hours or less, or 6 hours or less.

[0116] Following the precipitation step, a separation step may be performed to separate the precipitate containing extracellular vesicles. The precipitate can be recovered by known solid-liquid separation methods. Examples of solid-liquid separation methods include centrifugation.

[0117] After the separation step, a dispersion step may be performed in which the recovered precipitate is dispersed in an aqueous medium. Examples of aqueous mediums include water, phosphate-buffered saline (PBS(-), etc.), HEPES, and Good's buffer such as Tris.

[0118] <Analysis process> The method according to this embodiment may further include an analytical step of analyzing the recovered extracellular vesicles.

[0119] The analytical process involves analyzing proteins, miRNAs, and extracellular vesicle markers. Examples of extracellular vesicle markers include CD81, CD63, CD9, Alix, and Tsg101. Preferred extracellular vesicle markers in the analytical process are CD81, CD63, and CD9.

[0120] The method according to this embodiment may include, as an analytical step, the step of measuring the amount of microRNA in the extracellular vesicles relative to the amount of protein in the fraction of recovered extracellular vesicles (hereinafter referred to as "miRNA / protein"). In this specification, miRNA / protein refers to the mass of miRNA per 1 μg of protein (ng). A higher miRNA / protein value indicates that the extracellular vesicles produced have a higher microRNA content and a lower amount of impurities. The amount of protein in the fraction of extracellular vesicles includes the amount of protein that makes up the extracellular vesicles, but since this amount is extremely small, this protein amount can be approximated as the amount of impurities. The amount of protein and microRNA in the fraction of extracellular vesicles can be measured by the method described in the examples described later.

[0121] The miRNA / protein ratio may be 1.5 or greater, or 3 or greater, and may be 50 or less, 30 or less, or 10 or less.

[0122] The method according to this embodiment may include, as an analytical step, the step of measuring the amount of microRNA in extracellular vesicles relative to the amount of extracellular vesicle markers. Since extracellular vesicles can contain miRNAs that function as early diagnostic markers for cancer and the like, it is necessary to recover extracellular vesicles that contain a large amount of miRNA. The amount of microRNA in extracellular vesicles relative to the amount of extracellular vesicle markers can be used as an indicator of how much microRNA is present in the obtained extracellular vesicles. The amount of extracellular vesicle markers and microRNA can be measured by the method described in the examples described later.

[0123] The amount of microRNA relative to the amount of extracellular vesicle markers may be the amount of microRNA relative to CD63 (hereinafter referred to as "miRNA / CD63") or the amount of microRNA relative to CD81 (hereinafter referred to as "miRNA / CD81"). In this specification, miRNA / CD63 refers to the mass (ng) of miRNA per 1 pg of CD63. In this specification, miRNA / CD81 refers to the mass (ng) of miRNA per 1 pg of CD81.

[0124] miRNA / CD63 may be 0.001 or greater, 0.01 or greater, or 0.1 or greater, and may be 5 or less, 3 or less, or 1 or less.

[0125] miRNA / CD81 may be 0.0001 or greater, 0.0005 or greater, 0.001 or greater, or 0.01 or greater, and may be 0.1 or less, 0.05 or less, or 0.03 or less.

[0126] The extracellular vesicles obtained by the method according to this embodiment contain a large amount of microRNA and can therefore be suitably used for the diagnosis of diseases such as cancer. [Examples]

[0127] The contents of this disclosure will be described in more detail below using examples and comparative examples, but this disclosure is not limited to the following examples. In the examples, "room temperature" means 20 to 30°C.

[0128] [miRNA level measurement] Total microRNA amount is Qubit TM Measurements were performed using the microRNA Assay Kit (Invitrogen) according to the kit protocol. MicroRNA Reagent was diluted 200-fold with microRNA Buffer. This dilution (sample before separation of extracellular vesicles: 190 μL, sample after purification of extracellular vesicles: 198 μL) was mixed with exosome solution (sample before separation of extracellular vesicles: 10 μL, sample after purification of extracellular vesicles: 2 μL), vortexed for 3-5 seconds, and allowed to stand at room temperature for 2 minutes. The fluorescence intensity of the solution was measured using a Qubit® 4.0 Fluorometer (Invitrogen) or a DeNovix DS-11 FX+.

[0129] [Protein content measurement] Micro BCA TM The Protein Assay Kit (Invitrogen) was used, and measurements were performed according to the kit protocol. A 2.0 mg / mL albumin (BSA) standard solution was diluted with D-PBS(-) to prepare standard solutions at concentrations of 200, 40, 20, 10, 5, 2.5, 1, and 0.5 μg / mL. Next, Micro BCA Reagents A, B, and C were mixed in a volume ratio of 25:24:1 to prepare Working Reagent (WR). Each exosome solution was diluted with D-PBS(-) (100-fold for samples before separation of extracellular vesicles; 20-fold for purified samples from Example 1 and Comparative Example 1; 50-fold for purified samples from Examples 2 and 3 and Comparative Examples 2 and 3). 100 μL of WR was added to each well of a 96-well plate, followed by 100 μL of diluted exosome solution. The wells were sealed and incubated at 60°C for 1 hour. The absorbance at 560 nm in each well was measured using a plate reader, Multiskan FC (Thermo Fisher).

[0130] [Measurement of CD63 quantity] Quantification was performed using the CD9 / CD63 ELISA kit for human exosome quantification (Cosmo Bio Co., Ltd.). First, the anti-CD9 antibody-immobilized plate and reagents were allowed to return to room temperature. The standard protein (2000 pg / mL) in the kit was diluted with assay buffer to prepare standard solutions at 200, 100, 50, 25, 12.5, 6.25, and 3.125 pg / mL. Sample preparation was then carried out. Each sample, including the purified sample, was diluted with assay buffer (sample before separation of extracellular vesicles: 100, 200, 400x; purified samples from Example 1 and Comparative Example 1: 5, 10x; Example 3 and Comparative Example 3: 200, 400x; Example 2 and Comparative Example 2: 400, 800x). 100 μL of the diluted standard protein or sample solution was added to each well of the plate. The plate was sealed and mixed with a plate shaker (800 rpm, 30 seconds), then allowed to stand at room temperature for 2 hours. The reaction solution was completely removed, and 300 μL of wash buffer was added to each well for washing. This procedure was repeated three times. 100 μL of HRP-labeled anti-CD63 antibody, diluted 500-fold with assay buffer, was added to each well, the plate was sealed, and mixed with a plate shaker. The reaction was allowed to stand at room temperature for 2 hours. The antibody solution was completely removed, and 300 μL of wash buffer was added to each well for washing. This procedure was repeated three times. 100 μL of substrate solution was added to each well, and the reaction was allowed to stand at room temperature for 20 minutes. After checking the color intensity, 50 μL of stop solution was added to each well. The absorbance of each well was measured using a Multiskan FC (Thermo Fisher) plate reader (measurement wavelength 450 nm).

[0131] [Measurement of CD81 levels] Quantitative analysis was performed using the CD81-Capture Human Exosome ELISA Kit (Streptavidin HRP) (Fujifilm Wako Pure Chemical Corporation). Each well of the Anti-CD81 Antibody-immobilized 96-Well Plate (strips used) was washed three times with 300 μL of a washing solution diluted 10-fold with MilliQ water, and the remaining liquid in the wells was removed. The samples were diluted with Sample Reaction Buffer (sample before separation of extracellular vesicles; 10,000-fold; test using magnetic particle dispersion used in Example 1; 100-fold; test using magnetic particles with Tim protein conjugates (Fujifilm Wako Pure Chemical Corporation, product name MagCapturePS); 10,000-fold; test using polymer flocculant from Thermo Fisher; 100,000-fold). Reference sample dilutions were prepared by progressively diluting the extracellular vesicle internal reference (293T UF) with Sample Reaction Buffer. Sample diluent, reference sample diluent, and Sample Reaction Buffer (as a blank) were dispensed into each well in 100 μL portions. A plate seal was applied, and the mixture was reacted at room temperature for 2 hours while stirring at approximately 500 rpm using a microplate shaker. After the reaction was complete, the reaction mixture was discarded, and each well was washed three times with 300 μL of (1×) washing solution to remove any remaining liquid. Next, a biotinylated antibody (Anti-CD81) was diluted 100-fold with Antibody Reaction Buffer to prepare the biotinylated anti-CD81 antibody reaction mixture. 100 μL of the biotinylated anti-CD81 antibody reaction mixture was dispensed into each well in 100 μL portions. A plate seal was applied, and the mixture was reacted at room temperature for 1 hour while stirring at approximately 500 rpm using a microplate shaker. After the reaction was complete, the reaction mixture was discarded, and each well was washed three times with 300 μL of (1×) washing solution to remove any remaining liquid. HRP-conjugated streptavidin was diluted 100-fold with Antibody Reaction Buffer and thoroughly mixed to prepare the HRP-labeled streptavidin reaction solution. 100 μL of the HRP-labeled streptavidin reaction solution was dispensed into each well.The plate was sealed and the reaction was allowed to proceed at room temperature for 2 hours while stirring at approximately 500 rpm using a microplate shaker. After the reaction was complete, the reaction solution was discarded, and each well was washed four times with 300 μL of (1×) washing solution. Then, the plate was inverted on a stack of paper towels or similar material and gently tapped to remove any remaining liquid from the wells. 100 μL of TMB Solution, returned to room temperature, was dispensed into each well and stirred for approximately 1 minute using a microplate shaker. The plate was sealed and allowed to stand at room temperature for 30 minutes. 100 μL of Stop Solution, returned to room temperature, was added to each well. After stirring for approximately 5 seconds using a microplate shaker, the absorbance at 450 nm and the absorbance at the secondary wavelength of 620 nm were immediately measured using a 96-well microplate reader Infinite 200 (TECAN).

[0132] [Example 1] HEK293T cells were cultured in DMEM (containing FBS(+)) at 37°C for 2 days (preliminary culture step). Next, the culture medium was changed to FreeStyle® 293 expression medium (hereinafter also referred to as "FreeStyle medium"), and the cells were cultured again in FreeStyle medium at 37°C for 2 days to collect the culture medium containing extracellular vesicles (culture step). Subsequently, the culture medium was centrifuged at 300×g for 5 minutes, 1200×g for 20 minutes, and 10000×g for 30 minutes at 4°C to remove coarse aggregates. After centrifugation, the culture supernatant was filtered through a 0.22 μm filter and then concentrated 33-fold using a 100 kDa ultrafiltration membrane (concentration step).

[0133] To 1 mL of concentrated culture supernatant, 1 mL of magnetic particle dispersion was added to adsorb extracellular vesicles onto the surface of the magnetic particles (adsorption step). After the adsorption step, the magnetic particles were separated from the liquid using a magnet (separation step). After the separation step, 500 μL of chelating agent aqueous solution was added to the magnetic particles to desorb extracellular vesicles from the surface of the magnetic particles, and the extracellular vesicles were purified (desorption step). The purification results are shown in Tables 1 and 2. The chelating agent aqueous solution used was a 500 mmol / L EDTA aqueous solution (pH 8.0) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) diluted with PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to an EDTA concentration of 20 mmol / L.

[0134] The purified extracellular vesicles contained 151.5 ng of miRNA, indicating a high miRNA content. Furthermore, the protein content was 39.5 μg, and the miRNA / protein ratio was 3.83, which is also high. A high miRNA / protein ratio suggests that the extracellular vesicles recovered had a high miRNA content and few impurities.

[0135] [Example 2] Instead of separating extracellular vesicles using magnetic particles as in Example 1, extracellular vesicles were separated by the following method, and the extracellular vesicles were recovered in the same manner as in Example 1.

[0136] To 1 mL of concentrated culture medium, 0.5 mL of a polymer flocculant (Thermo Fisher, product name Total Exosome Isolation) was added and mixed, then allowed to stand overnight at 4°C (precipitation step). The resulting precipitate was separated by centrifugation (separation step). 100 μL of PBS(-) was added to redisperse the precipitate (redispersion step).

[0137] The miRNA content in the recovered extracellular vesicles was 951.7 ng, indicating a high miRNA concentration. Additionally, the protein content was 200 μg, and the miRNA / protein ratio was 4.75, indicating a high miRNA / protein ratio.

[0138] [Example 3] Instead of separating extracellular vesicles using magnetic particles as in Example 1, extracellular vesicles were separated by the following method, and the extracellular vesicles were recovered in the same manner as in Example 1.

[0139] Extracellular vesicles were separated into 100 μL of eluate using magnetic particles containing Tim protein conjugates (manufactured by Fujifilm Wako Pure Chemical Industries, product name MagCapturePS) in 1 mL of concentrated culture medium.

[0140] The miRNA content in the recovered extracellular vesicles was 1.3 ng, indicating a low miRNA level. Additionally, the protein content was 2.8 μg, and the miRNA / protein ratio was 0.46, indicating a low miRNA / protein ratio.

[0141] [Example 4] Instead of separating extracellular vesicles using magnetic particles as in Example 1, extracellular vesicles were separated by the following method, and the extracellular vesicles were purified in the same manner as in Example 1.

[0142] To 1 mL of concentrated culture supernatant, 1 mL of a white solid dispersion was added to adsorb extracellular vesicles onto the surface of the white solid (adsorption step). After the adsorption step, the white solid was centrifuged to separate the liquid from the white solid (separation step). After the separation step, 500 μL of chelating agent aqueous solution was added to the white solid to desorb the extracellular vesicles from the surface of the white solid, and the extracellular vesicles were purified (desorption step). The chelating agent aqueous solution used was a 500 mmol / L EDTA aqueous solution (pH 8.0) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) diluted with PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to an EDTA concentration of 20 mmol / L.

[0143] The miRNA content in the recovered extracellular vesicles was 254.5 ng, indicating a high miRNA concentration. Additionally, the protein content was 18 μg, and the miRNA / protein ratio was 14.14, indicating a high miRNA / protein ratio.

[0144] [Comparative Example 1] Instead of the FreeStyle® 293 expression medium used in Example 1, the culture medium containing extracellular vesicles was collected using DMEM (without FBS(+)), and the extracellular vesicles were collected in the same manner as in Example 1.

[0145] The miRNA content in the recovered extracellular vesicles was 1.19 ng, indicating a low miRNA level. Additionally, the protein content was 1.15 μg, and the miRNA / protein ratio was 1.03, indicating a low miRNA / protein ratio.

[0146] [Comparative Example 2] Instead of the FreeStyle® 293 expression medium used in Example 2, the culture medium containing extracellular vesicles was collected using DMEM, and the extracellular vesicles were collected in the same manner as in Example 2.

[0147] The miRNA content in the recovered extracellular vesicles was 1.64 ng, indicating a low miRNA level. Additionally, the protein content was 3.18 μg, and the miRNA / protein ratio was 0.51, indicating a low miRNA / protein ratio.

[0148] [Comparative Example 3] Instead of the FreeStyle® 293 expression medium used in Example 3, the culture medium containing extracellular vesicles was collected using DMEM, and the extracellular vesicles were collected in the same manner as in Example 3.

[0149] The miRNAs contained in the recovered extracellular vesicles were below the detection limit.

[0150] [Reference example 1] The magnetic particle dispersion used in Example 1 was prepared by the following method.

[0151] Process (i) (fine magnetic material adsorption) 1 g of polydivinylbenzene particles (particle size 2.2 μm) were dispersed in 50 mL of 0.1 M sodium chloride pure water at room temperature at a rotation speed of 100 rpm. To this particle dispersion, 1.2 g of a magnetic material (material: magnetite, particle size: 10 nm, product name: EMG607, manufactured by Ferrotec Corporation) having a cationic dispersant on its surface was added, and the mixture was reacted at room temperature at a rotation speed of 100 rpm for 2 hours. The solid components (particles) were filtered off and washed with pure water.

[0152] Process (ii) (Drying and high-speed airflow treatment) The particles washed with pure water were dried at 50°C for 15 hours to obtain 2.18 g of magnetized dried particles. These magnetized particles were placed in a hybridization system NHS-0 (manufactured by Nara Machine Works Co., Ltd.) at a rotation speed of 11300 min⁻¹. -1 The treatment was performed for 5 minutes at an average temperature of 35°C during processing.

[0153] Process (iii) (Coating of magnetized particles with metal oxide) One g of the magnetized particles was dispersed in nine mL of 2-propanol, and 0.4 g of tetraethyl orthosilicate was added. The mixture was stirred at room temperature at 180 rpm. 0.35 mL of 25% aqueous ammonia was added, and the mixture was reacted at 60°C at 180 rpm for four hours. After washing with methanol and drying under reduced pressure, the cross-sectional shape of the particles was examined using a transmission electron microscope. The thickness of the metal oxide layer was approximately 0.02 to 0.05 μm.

[0154] Step (iv) (Preparation of magnetic particle dispersion) Stock solutions were prepared at 10 times the final concentration of each component, so that the final concentrations were 3700 mg / L of sodium bicarbonate, 109 mg / L of disodium hydrogen phosphate, and 200 mg / L of calcium chloride. After mixing these solutions, the concentrations were adjusted by dilution with pure water. Magnetic particles were added so that the concentration of magnetic particles in the magnetic particle dispersion was 10 mg / mL.

[0155] The final concentrations of sodium bicarbonate, disodium hydrogen phosphate, and calcium chloride salts, 3700 mg / L, 109 mg / L, and 200 mg / L respectively, represent the concentrations of each salt based on the volume of the magnetic particle dispersion.

[0156] [Reference example 2] The white solid dispersion used in Example 4 was prepared by the following method.

[0157] Each component was dissolved and mixed in pure water to achieve final concentrations of 37,000 mg / L sodium bicarbonate, 1,090 mg / L disodium hydrogen phosphate, and 2,000 mg / L calcium chloride. After mixing, the mixture was left to stand at room temperature overnight, allowing the white solid to settle.

[0158] [Table 1]

[0159] [Table 2]

Claims

1. The process includes a culture step in which cells are cultured in a culture medium to obtain a culture supernatant containing extracellular vesicles, A method for producing extracellular vesicles, wherein the culture medium is FreeStyle™ 293 expression medium.

2. A step of bringing the culture supernatant into contact with a solid support having a metal oxide on its surface in the presence of carbonate ions, phosphate ions, and calcium ions, thereby adsorbing the extracellular vesicles onto the solid support. A step of separating the solid support from the liquid containing the culture supernatant, and A method for producing extracellular vesicles according to claim 1, further comprising the step of desorbing the extracellular vesicles from the solid phase carrier.

3. The method for producing extracellular vesicles according to claim 1, further comprising the step of mixing a polymer flocculant with the culture supernatant to precipitate the extracellular vesicles.

4. A step of contacting the culture supernatant with a solid support containing phosphorus and calcium to adsorb the extracellular vesicles onto the solid support. A step of separating the solid support from the liquid containing the culture supernatant, and A method for producing extracellular vesicles according to claim 1, further comprising the step of desorbing the extracellular vesicles from the solid phase carrier.

5. A method for producing extracellular vesicles according to any one of claims 1 to 4, further comprising a concentration step of concentrating the culture supernatant by ultrafiltration.

6. The method for producing extracellular vesicles according to claim 5, wherein the concentration step is a step of concentrating components that have not passed through the ultrafiltration membrane using an ultrafiltration membrane with a fractional molecular weight cutoff of 50 to 200 kDa.

7. A method for producing extracellular vesicles according to any one of claims 1 to 4, further comprising the step of measuring the amount of microRNA in the extracellular vesicles relative to the amount of protein in the fraction of the recovered extracellular vesicles.

8. A method for producing extracellular vesicles according to any one of claims 1 to 4, further comprising the step of measuring the amount of microRNA in the extracellular vesicles relative to the amount of extracellular vesicle markers, using the recovered extracellular vesicles as a measurement sample.

9. A method for producing extracellular vesicles according to any one of claims 1 to 4, wherein the cells are human embryonic kidney cells.

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