Method for producing extracellular vesicle derived from plant, mushroom, or microalgae

Abstract

The present invention provides a method for producing extracellular vesicles, the method including a step for passing a crushed liquid of plants, mushrooms, or microalgae through a depth filter, and a step for subjecting the crushed liquid after passing through the depth filter to ultrafiltration treatment, the depth filter being composed of filaments.

Description

Method for producing extracellular vesicles derived from plants, mushrooms, or microalgae

[0001] The present invention relates to a method for producing extracellular vesicles derived from plants, mushrooms, or microalgae.

[0002] Extracellular vesicles (EVs) are a collective term for lipid bilayer-enclosed vesicles secreted from cells in various organisms, including animals and plants, and include exosomes derived from endosomal membranes. EVs store proteins, lipids, nucleic acids, and other substances. EVs are known to have various physiologically active effects (Patent Documents 1 to 5), and are therefore expected to be applied to medicine. Furthermore, there is a demand for an industrial method for producing EVs.

[0003] Known methods for producing animal-derived EVs include ultracentrifugation, TFF, and PEG precipitation (Non-Patent Document 1). Known methods for producing plant-derived EVs include ultracentrifugation (Patent Documents 1 to 4), TFF (Patent Document 6), and PEG precipitation (Patent Document 7). However, production methods that include a centrifugation step have low reproducibility, and therefore a production method with high reproducibility is desired.

[0004] Japanese Patent Publication No. 2018-531932 Japanese Patent Publication No. 2020-535189 Japanese Patent Application Laid-Open No. 2021-193070 Japanese Patent Publication No. 2022-525131 Japanese Patent Publication No. 2022-543061 Japanese Patent Publication No. 2022-539228 International Publication No. 2016 / 185564

[0005] SIDHOM, Karim; OBI, Patience O.; SALEEM, Ayesha. A review of exosomal isolation methods: is size exclusion chromatography the best option?. International journal of molecular sciences, 2020, 21.18: 6466.

[0006] The problem to be solved by the present invention is to provide a novel and highly reproducible method for producing extracellular vesicles.

[0007] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered a novel method for producing extracellular vesicles from plants, mushrooms, or microalgae, and completed the present invention.

[0008] That is, the present invention is as follows: [1] A method for producing extracellular vesicles, comprising the steps of passing a disruption solution of plants, mushrooms, or microalgae through a depth filter and ultrafiltrating the disruption solution after passing through the depth filter, wherein the depth filter is made of filaments. [2] The method according to [1], wherein the step of passing the disruption solution through a depth filter is a step of passing the disruption solution through at least two types of depth filters having different pore sizes. [3] The method according to [2], wherein the at least two types of depth filters include depth filters having pore sizes of 10 to 300 μm and 0.5 to 75 μm. [4] A method for producing extracellular vesicles, comprising the steps of allowing a disruption solution derived from plants, mushrooms, or microalgae to stand and separating it into an aqueous layer and a contaminant layer, and separating the aqueous layer and ultrafiltrating the separated aqueous layer.

[0009] In [1] to [4], the method for producing extracellular vesicles may be a method for extracting extracellular vesicles, a method for purifying extracellular vesicles, or a method for producing extracellular vesicles.

[0010] The present invention provides a novel method for producing extracellular vesicles from plants, mushrooms, or microalgae.

[0011] The results of nanoparticle tracking analysis of parsley EVs obtained by centrifugation and the depth method are shown. The parsley EVs obtained by centrifugation and the depth method were applied to mouse macrophage cells, and the expression levels of inflammatory cytokines (IL-6 or IL-1β) were shown when LPS stimulation was applied. The results of RNA-seq analysis of gene expression changes when LPS stimulation was applied to mouse macrophage cells and parsley EVs obtained by centrifugation and the depth method were applied. Figure 1C shows the results of extracting only cytokines. The results of nanoparticle tracking analysis of parsley EVs obtained by centrifugation and the static method are shown. The results of LPS stimulation were applied to mouse macrophage cells and the expression levels of inflammatory cytokines (IL-6 or IL-1β) were shown.

[0012] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. However, the present invention is not limited to the following embodiment.

[0013] The method of this embodiment is a method for producing extracellular vesicles.

[0014] Extracellular vesicles (EVs) are a general term for vesicles surrounded by a lipid bilayer that are secreted from cells of various organisms, such as animals and plants, and include exosomes derived from endosomal membranes. In this specification, extracellular vesicles are also referred to as EVs, and EVs has the same meaning as extracellular vesicles. In this embodiment, extracellular vesicles can be obtained by a method using a depth filter and ultrafiltration, or a method including a standing step and ultrafiltration.

[0015] Depth Filter-Ultrafiltration Process The method of this embodiment includes passing a solution of crushed plants, mushrooms, or microalgae through a depth filter.

[0016] The disruption solution may be obtained by disrupting plants, mushrooms, or microalgae using a known method. For example, disruption may be performed using a mixer or a millstone, or the disruption solution may be obtained by squeezing or other methods. In this embodiment, the disruption solution may be a solution obtained by disrupting plants, mushrooms, or microalgae. The disruption solution may be a solution obtained after removing impurities from the solution obtained by disrupting plants, mushrooms, or microalgae, for example, by natural settling, centrifugation, or filtration through cloth or gauze. When obtaining the disruption solution, the plants, mushrooms, or microalgae may be directly disrupted, or the plants, mushrooms, or microalgae may be disrupted by immersing them in a buffer solution such as PBS. The mixing ratio of the buffer solution such as PBS to the plants, mushrooms, or microalgae may be a mixing ratio that results in a disruption solution. For example, the mass of the buffer solution such as PBS may be 50 times or less, preferably 25 times or less, more preferably 15 times or less, and even more preferably 10 times or less, the mass of the plants, mushrooms, or microalgae.

[0017] The disruption liquid used in this embodiment is a disruption liquid of plants, mushrooms, or microalgae belonging to the Apiaceae, Brassicaceae, Asteraceae, Fabaceae, Liliaceae, Amaryllidaceae, Amaranthaceae, Cucurbitaceae, Zingiberaceae, Polygonaceae, Solanaceae, Dioscoreaceae, Poaceae, or Rutaceae families.

[0018] The disruption liquid may contain disruption liquids from two or more different plants selected from the same family, or may contain disruption liquids derived from two or more different types of mushrooms or microalgae. Furthermore, the disruption liquid may contain disruption liquids from one or more different plants selected from the same family and one or more plants selected from a family different from the family. It may contain disruption liquids from plants of two or more different families. It may contain disruption liquids from plants and mushrooms, from plants and microalgae, from mushrooms and microalgae, or from plants, mushrooms, and microalgae.

[0019] The plants may be grown outdoors or hydroponically. The mushrooms may be naturally grown or may be grown in an artificial medium or on felled logs. The microalgae may be cultivated.

[0020] Plants of the Apiaceae family include, but are not limited to, parsley, Violet Harmony, mitsuba, angelica tree, fruit carrot, carrot, dill, fennel, cilantro, and parsnip. Plants of the Brassicaceae family include, but are not limited to, Brussels sprouts, Romanesco, arugula, cauliflower, wasabi radish, komatsuna, wasabi, cauliflower, red radish, Selvatica, bok choy, broccoli, mizuna, radish, daikon radish, watercress, leaf mustard, turnip, and Chinese cabbage. Plants of the Asteraceae family include, but are not limited to, artichoke, butterbur, chrysanthemum, burdock, red kidney bean, lettuce, endive, Jerusalem artichoke, romaine lettuce, and red lettuce. Leguminous plants include, but are not limited to, edamame, yam, Moroccan beans, green beans, snap peas, pea sprouts, or snap beans. Liliaceae or Amaryllidaceae plants include, but are not limited to, chives, Belgian escharlotte, leeks, onions, escharlotte, asparagus, or spring onions. Amaranthaceae plants include, but are not limited to, epazote, amaranth, Swiss chard, spinach, beets, or hijiki seaweed. Cucurbitaceae plants include, but are not limited to, watermelon, zucchini, or loofah. Zingiberaceae plants include, but are not limited to, myoga ginger or Yanaka ginger. Polygonaceae plants include, but are not limited to, rhubarb. The Solanaceae plant is not particularly limited, but may be, for example, paprika, manganji pepper, potato, or bell pepper. The Yamaceae plant is not particularly limited, but may be, for example, Chinese yam. The Gramineae plant is not particularly limited, but may be, for example, baby corn or corn. The Rutaceae plant is not particularly limited, but may be, for example, kaffir lime leaves or kumquat. The microalgae is not particularly limited, but may be, for example, Euglena.The mushrooms are not particularly limited, but may be, for example, brown mushrooms, shiitake mushrooms, shimeji mushrooms, or enoki mushrooms.

[0021] The disruption liquid used in this embodiment is a disruption liquid for rosemary, marjuram, lemon palm, spearmint, thyme, oregano, peppermint, red shiso, sweet basil, sage, Brussels sprouts, Romanesco, arugula, cauliflower, wasabi radish, komatsuna, wasabi, cauliflower, red radish, selvatica, bok choy, broccoli, mizuna, radish, daikon, watercress, leaf mustard, turnip, tatsoi, artichoke, butterbur, chrysanthemum, burdock, red kidney bean, lettuce, endive, Jerusalem artichoke, romaine lettuce, radish, sunny lettuce, parsley, violet harmony, mitsuba, angelica tree, fruit carrot This product contains crushed liquid derived from: carrots, dill, fennel, coriander, parsnips, edamame beans, cucumber beans, Moroccan green beans, green peas, snap peas, pea sprouts, snap beans, chives, écharlotte, Belgian écharlotte, leeks, onions, asparagus, leeks, epazote, amaranth, Swiss chard, spinach, beets, hijiki seaweed, watermelon, zucchini, loofah, myoga ginger, Yanaka ginger, rhubarb, paprika, manganji peppers, potatoes, bell peppers, nagaimo, baby corn, corn, kaffir lime leaves, kumquats, brown mushrooms, shiitake mushrooms, shimeji mushrooms, enoki mushrooms, or Euglena.

[0022] The disruption liquid used in this embodiment is preferably a disruption liquid derived from Brussels sprouts, parsley, or Euglena.

[0023] "Depth filter" refers to a series of successively arranged filters with decreasing pore size used to remove contaminants from the disruption fluid. The three-dimensional matrix of the depth filter forms a labyrinth-like pathway through which the disruption fluid passes, mechanically trapping contaminants throughout the depth of the matrix.

[0024] The depth filter used in this embodiment is a filament-shaped depth filter. A filament-shaped depth filter refers to a depth filter made of filaments produced, for example, by a melt-blowing method or a spunbonding method. The filaments may contain, as a main component, at least one selected from the group consisting of polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate, polybutylene terephthalate, and polycyclohexylene dimethylene terephthalate, polyamides such as nylon, ethylene-vinyl alcohol copolymers, and polyvinyl alcohol and polyacrylonitrile. The term "main component" refers to the component with the highest content. Commercially available depth filters may be used, such as CCP-30-C1H (pore size: 30 μm), CCP-10-C1H (pore size: 10 μm), CCP-7-C1H (pore size: 7 μm), CCP-3-C1H (pore size: 3 μm), CCP-1-C1H (pore size: 1 μm), TCW-150N-PPS (pore size: 150 μm), TCW-100N-PPS (pore size: 100 μm), TCW-75N-PPS (pore size: 75 μm), TCW-50N-PPS (pore size: 50 μm), and TCW-25N-PPS (pore size: 100 μm). size: 25μm), TCW-10N-PPS (pore size: 10μm), TCW-5N-PPS (pore size: 5μm), TCW-3N-PPS (pore size: 3μm), TCW-1N-PPS (pore size: 3μm) size: 1 μm), TCW-08N-PPS (pore size: 0.8 μm), and TCW-05N-PPS (pore size: 0.5 μm) (manufactured by Advantec).

[0025] The disruption solution may be treated using depth filters with two or more different pore sizes. The pore size of the depth filter may be selected from 0.5 μm to 300 μm. For example, when two types of depth filters are used, the pore size of the first depth filter is selected from 10 μm to 300 μm, preferably 50 μm to 200 μm, and more preferably 100 μm to 150 μm, and the pore size of the second depth filter is selected from 0.5 μm to 75 μm, preferably 0.5 μm to 50 μm, and more preferably 0.5 μm to 5 μm. When depth filters with two or more different pore sizes are used, it is preferable to pass the disruption solution through the depth filter with the largest pore size to the depth filter with the smallest pore size. In this specification, "X to Y" (X and Y represent numerical values) means a range of X or more and Y or less, unless otherwise specified.

[0026] When depth filters having two or more different pore sizes are used, the combination of depth filter pore sizes may be selected based on the turbidity of the disruption solution after passing through the depth filter. The turbidity may be measured by a known method, and the turbidity of the disruption solution after passing through the depth filter is preferably 300 FTU (Formazin Turbidity Unit (JIS K0101)) or less, more preferably 150 FTU or less, and even more preferably 100 FTU or less.

[0027] The disruption solution may be passed through a depth filter without dilution, or may be diluted and passed through a depth filter. The solvent used for dilution may be a known solvent for filtration through a depth filter. For example, the mass of the solvent may be 50 times or less, preferably 25 times or less, more preferably 15 times or less, and even more preferably 10 times or less, the mass of the disruption solution.

[0028] The step of passing the disruption solution through a depth filter is not particularly limited, but may be carried out, for example, at 25°C or lower, preferably 15°C or lower, more preferably 10°C or lower, and even more preferably 5°C or lower.

[0029] The pressure applied to the disruption solution passing through the depth filter is not particularly limited, but is preferably controlled from the viewpoint of increasing the processing speed. For example, the pressure difference before and after the depth filter is preferably 0 to 0.49 MPa (excluding 0), more preferably 0.05 to 0.30 MPa, and even more preferably 0.10 to 0.20 MPa. The pressure can be controlled by any known method, such as a method utilizing a height difference or a method utilizing a pump.

[0030] The method of this embodiment further includes a step of ultrafiltration of the disruption solution after passing through the depth filter. The ultrafiltration may be performed using two or more types of ultrafiltration membranes. Commercially available ultrafiltration membranes may be used, such as TFF Omega membranes (manufactured by Cytiva). An ultrafiltration membrane may be selected depending on the size of the target extracellular vesicles. For example, the nominal molecular weight cutoff of the ultrafiltration membrane may be selected from 1 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa, 100 kDa, 300 kDa, 500 kDa, and 1000 kDa, preferably in the range of 50 kDa to 1000 kDa, and more preferably in the range of 100 kDa to 500 kDa.

[0031] The ultrafiltration step may include pretreatment, which may be carried out by a known method, preferably by passing the solution through a membrane depth filter. The depth filter for pretreatment may be a commercially available one, such as SC050PDH4 (pore size: 0.5 μm to 15 μm), SC050PDK5 (pore size: 1.5 μm to 20 μm), SC050PDP8 (pore size: 6 μm to 30 μm), SC050P100 (pore size: 1 μm to 3 μm), SC050P200 (pore size: 3 μm to 6 μm), SC050P250 (pore size: 4 μm to 9 μm), SC050P700 (pore size: 6 μm to 15 μm), SC050P900 (pore size: 8 μm to 20 μm), and SC050B020 (pore size: 1 μm to 20 μm). For example, SC050PDD1 (pore size: 0.1 μm to 0.85 μm), SC050PDE2 (pore size: 0.2 μm to 3.5 μm) (manufactured by Cytiva) may be used.

[0032] The ultrafiltration membrane is not particularly limited, but may contain, as a main component, for example, polyethylene, polypropylene, polyvinylidene fluoride, ethylene-vinyl alcohol copolymer, polyamide, polyimide, polyetherimide, polystyrene, polysulfone, polyvinyl alcohol, polyphenylene ether, polyphenylene sulfide, cellulose acetate, polyacrylonitrile, polytetrafluoroethylene (PTFE), or the like.

[0033] Ultrafiltration is not particularly limited as long as it is a method using an ultrafiltration membrane, and may be performed by any known method. For example, ultrafiltration may be a method of forced filtration by pressure operation or centrifugation operation, or a method generally referred to as "diafiltration," which performs filtration by passive diffusion. Alternatively, it may be a method of introducing a solution into an ultrafiltration membrane at a constant flow rate. When performing ultrafiltration, the disrupted tissue solution after passing through a depth filter may be filtered as is. The mixing ratio of the disrupted tissue solution to a solvent such as water is not particularly limited, and may be any ratio that allows ultrafiltration to be performed.

[0034] The ultrafiltration treatment is not particularly limited, but may be carried out, for example, at 25°C or lower, preferably 15°C or lower, more preferably 10°C or lower, and even more preferably 5°C or lower.

[0035] The ultrafiltration treatment may be carried out until the volume of the liquid before filtration is reduced to at least two times, preferably at least five times, more preferably at least ten times the volume of the liquid after filtration.

[0036] In this embodiment, the ultrafiltration devices may be connected in parallel to increase the amount of water that can be processed at one time.

[0037] In this embodiment, the step of passing the crushed liquid of plants, mushrooms, or microalgae through a depth filter and the step of ultrafiltration of the crushed liquid after passing through the depth filter may be performed consecutively.

[0038] The size of extracellular vesicles obtained from plants, mushrooms, or microalgae can be analyzed by methods known to those skilled in the art. For example, the particle size, particle size distribution, and concentration of extracellular vesicles can be examined by nanoparticle tracking analysis (NTA).

[0039] The extracellular vesicles obtained using depth filters and ultrafiltration may have a particle diameter of, for example, 10 to 300 nm, and the concentration of extracellular vesicles may be high at particle diameters of 50 to 250 nm.

[0040] Settling-Ultrafiltration Treatment The method of this embodiment may include a step of settling the disrupted liquid derived from plants, mushrooms, or microalgae to separate it into a water layer and a contaminant layer.

[0041] The disruption solution may be obtained in the same manner as described above in the depth filter-ultrafiltration process.

[0042] The plants, mushrooms, or microalgae may be selected in the same manner as the plants, mushrooms, or microalgae described above in the depth filter-ultrafiltration process.

[0043] The method may further include a step of passing the disruption solution through a filter mesh prior to the step of allowing the disruption solution to stand. The filter mesh is not particularly limited as long as it can be sterilized, and examples thereof include nylon mesh and metal mesh made of metal such as stainless steel. The mesh size of the filter mesh is 200 μm or less, preferably 150 μm or less, and more preferably 100 μm or less.

[0044] The step of allowing the disruption solution to stand is not particularly limited, and may be carried out at a temperature of, for example, 0 to 20° C., preferably 1 to 10° C., and more preferably 2 to 5° C. The time for allowing the solution to stand is, for example, 6 hours or more, preferably 8 hours or more, more preferably 10 hours or more, and even more preferably 12 hours or more.

[0045] The method of this embodiment may further include the steps of separating the aqueous layer and subjecting the separated aqueous layer to ultrafiltration.

[0046] The aqueous layer may be recovered by a known method.

[0047] The method of ultrafiltration may be carried out in the same manner as described above for the depth filter-ultrafiltration process.

[0048] In this embodiment, the steps of leaving the crushed liquid derived from plants, mushrooms, or microalgae to stand and separating it into an aqueous layer and an impurity layer, and the steps of separating the aqueous layer and subjecting the separated aqueous layer to ultrafiltration may be performed consecutively.

[0049] In this embodiment, the method may further include a step of pretreating the aqueous layer prior to the ultrafiltration step. The pretreatment may be performed by a known method, and may include, for example, a step of passing the aqueous layer through a depth filter.

[0050] The extracellular vesicles obtained using the settling method and ultrafiltration may have a particle diameter of, for example, 10 to 700 nm, and the concentration of extracellular vesicles may be high at particle diameters of 50 to 600 nm. Extracellular vesicles having a more limited particle size range may be obtained using a depth filter and ultrafiltration than using a settling method and ultrafiltration.

[0051] The extracellular vesicles obtained by any of the methods of this embodiment may be sterilized by passing them through a membrane filter. The pore size of the membrane filter may be, for example, 0.45 μm or 0.2 μm.

[0052] The extracellular vesicles obtained by any of the methods of the present embodiment may be dried using a freeze-drying method, which involves a drying step at cryogenic or low temperatures (approximately -200°C to -30°C). Freeze-drying is also called vacuum lyophilization, and is a method in which the material to be dried is cooled with a refrigerant and the solvent is removed by sublimation under vacuum. Common refrigerants used in freeze-drying include a mixture of dry ice and methanol (-78°C) and liquid nitrogen (-196°C). Impurities may be further removed from the extracellular vesicles obtained by any of the methods of the present embodiment. For example, impurities may be removed using an ion exchange membrane, and an anion exchange membrane is preferably used.

[0053] The present invention will be described in more detail below with reference to specific examples, although the present invention is not limited thereto.

[0054] (1) Functional Evaluation of Parsley EVs Prepared by Different Methods (1-1) Preparation of EVs Using Centrifugation Parsley was washed with water in a plastic bucket, drained, and then juiced using a household juicer (HUROM). The juice was centrifuged at 3,000 g for 20 minutes and then at 10,000 g for 40 minutes using an Avati J-E (JA-14 rotor, Bechman Courlter) to remove large debris. The resulting supernatant was then pretreated through a membrane depth filter (PDP8, PDE2, Cytiva) and then ultrafiltered using a TFF Omega membrane (Cytiva, nominal molecular weight cutoff: 100 kDa or 500 kDa) to obtain an EV sample.

[0055] (1-2) Preparation of EVs Using a Depth Filter Parsley was washed with water in a plastic bucket, drained, and then juiced using a household juicer (manufactured by HUROM). This juice was then passed through filamentous depth filters CCP-30-C1H and CCP-1-C1H (manufactured by Advantec) at a flow rate of 2.9 L / min, followed by pretreatment using membrane depth filters (PDP8, PDE2, manufactured by Cytiva) similar to those used in the centrifugation method. The pretreated solution was subjected to ultrafiltration using a TFF Omega membrane (manufactured by Cytiva, nominal molecular weight cutoff: 100 kDa or 500 kDa) to obtain an EVs sample.

[0056] (1-3) Preparation of EVs using the Settling Method Parsley was washed with water in a plastic bucket, drained, and then juiced using a household juicer (manufactured by HUROM). This juice was passed through a 132 μm filter mesh and allowed to stand overnight at 4°C. The next day, it was confirmed that larger fragments had settled, and the supernatant was used alone for pretreatment using a membrane depth filter (PDP8, PDE2, manufactured by Cytiva) similar to that used in the centrifugation method. Then, an EV sample was obtained by ultrafiltration using a TFF Omega membrane (manufactured by Cytiva, nominal molecular weight cutoff: 100 kDa or 500 kDa).

[0057] (1-4) Nanoparticle Tracking Analysis (NTA) The particle size, particle size distribution, and concentration of extracellular vesicles obtained by centrifugation, depth filtration, and static filtration were measured using a NanoSight LM10 (Malvern Instrument) equipped with a 405 nm blue laser. Figure 1A shows the results for EV samples prepared by centrifugation and depth filtration, and Figure 2A shows the results for EV samples prepared by centrifugation and static filtration. The results in Figures 1 and 2 indicate that samples prepared by centrifugation contained EVs with an average size of 156 nm (50-600 nm). Furthermore, samples prepared by the depth filtration method contained parsley EVs with an average size of 111 nm (50-250 nm), and samples prepared by static filtration contained EVs with an average size of 153 nm (50-600 nm). From the above, it was found that the depth method can prepare EVs of a more specific size than the centrifugation method, and that the static method can also prepare EVs of a similar size to those prepared by the centrifugation method.

[0058] (1-5) Culture of Mouse Macrophage Cells Mouse macrophage cells (Raw-Blue cells: manufactured by InvivoGen) were cultured in Dulbecco's Modified Eagle Medium (manufactured by Thermo Fisher) supplemented with 10% FBS, 100 μg / ml Normocin (manufactured by InvivoGen), and Pen-Strep (100 U / ml) at 37°C and 5% CO. ２ Cultured in an incubator, 4.7 x 10 ６ The cells were subcultured at a rate of 100 mm per 100 mm dish.

[0059] (1-6) LPS stimulation treatment Raw-Blue cells 0.8 x 10 ６ EVs were seeded in a 12-well dish at 2.5 x 10 cells / well. ９ and 1.0 x 10 １０After incubation with 1 μg / ml of LPS for 16 hours, the cells were washed with PBS and stimulated with 1 μg / ml LPS (derived from Salmonella minnesota R595; manufactured by WAKO) for 24 hours. After stimulation, the Raw-Blue cells were washed with PBS, and total RNA was extracted from the cells using TRIsure (manufactured by Nippon Genetics).

[0060] (1-7) Evaluation of the Effect of EVs on the Expression of Inflammatory Cytokines in LPS-Stimulated Mouse Macrophage Cells. The effect of EVs on the expression of inflammatory cytokines (IL-6 and IL-1β) in LPS-stimulated mouse macrophage cells was evaluated using the expression of inflammatory cytokines (IL-6 and IL-1β) in LPS-stimulated mouse macrophage cells as an index. The expression levels of IL-6 and IL-1β were measured by RT-qPCR using RNA extracted from LPS-stimulated mouse macrophage cells. Gapdh was used as a correction gene, and the values ​​were expressed as relative values ​​(fold change) when the expression level in cells stimulated with LPS, in the presence of PBS instead of EVs, was set to 1. The effect of EVs on the expression of inflammatory cytokines in LPS-stimulated mouse macrophage cells was evaluated by RNA-seq analysis. Using RNA extracted from LPS-stimulated mouse macrophage cells, an mRNA-seq library was prepared by polyA selection and sequenced using NavaSeq (Illumina). Sequencing was performed using a 150 bp paired-end method, obtaining 6 Gb of data (approximately 20 million reads). Gene expression matrices were generated using the Star (Spliced ​​transcripts alignment to a reference)-RSEM (RNA-Seq by Expectation-Maximization) method. Comparative gene expression analysis was performed using edgeR. Genes with an expression ratio of 0.6 or greater and a significance level (FDR) of 0.05 or less were extracted as genes with significantly fluctuating expression. The expression level of each gene for each sample was standardized by z-score and displayed as a heatmap.

[0061] Figure 1B shows the expression levels of IL-6 and IL-1β when mouse macrophage cells were exposed to EVs prepared by the centrifugation method and the depth method and stimulated with LPS, and Figure 2B shows the expression levels of IL-6 and IL-1β when mouse macrophage cells were exposed to EVs prepared by the centrifugation method and the static method and stimulated with LPS.

[0062] Figure 1C shows the results of RNA-seq analysis of gene expression changes after LPS stimulation in mouse macrophage cells treated with parsley EVs prepared by the depth method (LPS_depth_PcELNs). The results revealed that the expression levels of 1,042 genes were significantly reduced in mouse macrophage cells treated with parsley EVs prepared by the depth method (LPS_depth_PcELNs) compared to those treated with PBS (LPS). Furthermore, parsley EVs prepared by the depth method had a stronger inhibitory effect than parsley EVs prepared by the centrifugation method (LPS_cent_PcELNs). Figure 1D shows the results of cytokine selection from 1,042 genes. Parsley EVs prepared by the depth method were found to significantly suppress the expression of inflammatory cytokines such as CXCL2, CXCL3, and TGFB1 in addition to IL-6 and IL-1B.

[0063] The results in Figures 1 and 2 demonstrate that in mouse macrophage cells supplemented with parsley EVs prepared by centrifugation, the expression of inflammatory cytokines decreased depending on the amount of EVs added, compared to when PBS was added. In contrast, in mouse macrophage cells supplemented with the same amount of parsley EVs prepared by the depth method and the static method, the expression of inflammatory cytokines was further reduced. In other words, parsley EVs prepared by the depth method and the static method had stronger inhibitory ability than parsley EVs prepared by centrifugation.

Claims

1. A method for producing extracellular vesicles, comprising the steps of passing a lysate of a plant, mushroom, or microalgae through a depth filter, and ultrafiltration of the lysate after passing through the depth filter, wherein the depth filter is composed of filaments.

2. The method according to claim 1, wherein the step of passing the disruption solution through a depth filter is a step of passing the disruption solution through at least two types of depth filters having different pore sizes.

3. The method of claim 2, wherein the at least two depth filters comprise depth filters with pore sizes of 10-300 μm and 0.5-75 μm.

4. A method for producing extracellular vesicles, comprising the steps of allowing a crushed liquid derived from plants, mushrooms, or microalgae to stand and separate into an aqueous layer and an impurity layer, and separating the aqueous layer and subjecting the separated aqueous layer to ultrafiltration.

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