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

Abstract

To provide a method for producing extracellular vesicles derived from plants, mushrooms, or microalgae. [Solution] A method for producing extracellular vesicles, comprising the steps of passing a crushed liquid of plants, mushrooms, or microalgae through a depth filter, and ultrafiltration of the crushed liquid after passing through the depth filter, wherein the depth filter is composed of filaments.

Description

[Technical Field]

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

[0002] Extracellular vesicles (EVs) are a collective term for lipid bilayer-enclosed vesicles secreted by 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 industrial methods 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, so a highly reproducible production method is needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2018-531932 [Patent Document 2] Special Publication No. 2020-535189 [Patent Document 3] Patent Publication No. 2021-193070 [Patent Document 4] Special Publication No. 2022-525131 [Patent Document 5] Special Publication No. 2022-543061 [Patent Document 6] Special Publication No. 2022-539228 [Patent Document 7] International Publication No. 2016 / 185564 [Non-patent literature]

[0005] [Non-Patent Document 1] 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. Summary of the Invention [Problem to be solved by the invention]

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

[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] Passing the crushed solution of plants, mushrooms, or microalgae through a depth filter; and A step of ultrafiltration of the disrupted solution after passing through the depth filter. A method for producing extracellular vesicles, comprising: The method wherein the depth filter is comprised 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 step of allowing the crushed liquid derived from plants, mushrooms, or microalgae to stand and separate into a water layer and a contaminant layer; A step of separating the aqueous layer and subjecting the separated aqueous layer to ultrafiltration. A method for producing extracellular vesicles, comprising:

[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. [Effects of the Invention]

[0010] The present invention makes it possible to provide a novel method for producing extracellular vesicles from plants, mushrooms, or microalgae. [Brief explanation of the drawings]

[0011] [Figure 1A] The results of nanoparticle tracking analysis of parsley EVs obtained by centrifugation and depth spectroscopy are shown. [Figure 1B] Parsley EVs obtained by centrifugation and depth filtration were applied to mouse macrophage cells, and the expression levels of inflammatory cytokines (IL-6 or IL-1β) were measured when stimulated with LPS. [Figure 1C] Parsley EVs obtained by centrifugation and depth spectroscopy were applied to mouse macrophage cells, and changes in gene expression upon LPS stimulation were analyzed using RNA-seq. [Figure 1D] Figure 1C shows the results of extracting only cytokines. [Figure 2A] The results of nanoparticle tracking analysis of parsley EVs obtained by centrifugation and static methods are shown. [Figure 2B]Parsley EVs obtained by centrifugation and static incubation were applied to mouse macrophage cells, and the expression levels of inflammatory cytokines (IL-6 or IL-1β) were shown when stimulated with LPS. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, the embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. It should be noted that 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 in various organisms, including 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, the extracellular vesicles can be obtained by a method using a depth filter and ultrafiltration, or a method including a settling step and ultrafiltration.

[0015] Depth filter - Ultrafiltration process The method of this embodiment includes a step of passing a liquid obtained by crushing a plant, mushroom, or microalgae through a depth filter.

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

[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 liquid from two or more different plants selected from the same family, or may contain disruption liquid from two or more different mushrooms or microalgae. Furthermore, the disruption liquid may contain disruption liquid from one or more different plants selected from the same family and disruption liquid from one or more plants selected from a family different from the family. It may contain disruption liquid from plants of two or more different families. It may contain disruption liquid from plants and mushrooms, or from plants and microalgae, or 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] The Umbelliferae plant is not particularly limited, but may be, for example, parsley, Violet Harmony, Mitsuba, Angelica keiskei, fruit carrot, carrot, dill, fennel, coriander, or parsnip. The Brassicaceae plant is not particularly limited, but may be, for example, Brussels sprouts, Romanesco, arugula, cauliflower, wasabi radish, komatsuna, wasabi, cauliflower, red radish, Selvatica, bok choy, broccoli, mizuna, radish, daikon radish, watercress, leaf mustard, turnip, or tatsoi. The Asteraceae plant is not particularly limited, but may be, for example, artichoke, butterbur sprout, chrysanthemum, burdock, golden kidney leaf, lettuce, endive, Jerusalem artichoke, romaine lettuce, radicchio, or sunny lettuce. The leguminous plant is not particularly limited, but may be, for example, edamame, urizumame, Moroccan beans, silken peas, snap peas, pea sprouts, or snap beans. The plant of the Liliaceae or Amaryllidaceae family is not particularly limited, but may be, for example, Chinese chive, Belgian escharlotte, leek, onion, escharlotte, asparagus, or spring onion. The Amaranthaceae plant is not particularly limited, but may be, for example, epazote, amaranth, Swiss chard, spinach, beet, or hijiki seaweed. The Cucurbitaceae plant is not particularly limited, but may be, for example, a watermelon, a zucchini, or a loofah. The Zingiberaceae plant is not particularly limited, but may be, for example, myoga or Yanaka ginger. The plant of the Polygonaceae family is not particularly limited, but may be, for example, rhubarb. The Solanaceae plant is not particularly limited, but may be, for example, paprika, manganji pepper, potato, or bell pepper. The plant of the Dioscoreaceae family is not particularly limited, but may be, for example, Chinese yam. The grass 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 kumquats. 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. The filament-shaped depth filter refers to a depth filter made of filaments produced by, for example, a melt-blowing method or a spunbonding method. The filaments are Polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate, polybutylene terephthalate, and polycyclohexylene dimethylene terephthalate; Polyamides such as nylon, ethylene vinyl alcohol copolymer, and Polyvinyl alcohol and polyacrylonitrile The "major component" refers to the component that is contained in the greatest amount. 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), TCW-25N-PPS (pore size: 25 μm), TCW-10N-PPS (pore size: 10 μm), TCW-5N-PPS (pore size: 5 μm), and TCW-3N-PPS (pore size: 5 μm). TCW-1N-PPS (pore size: 3 μm), TCW-08N-PPS (pore size: 0.8 μm), and TCW-05N-PPS (pore size: 0.5 μm) (manufactured by Advantec) may be used.

[0025] The disruption solution may be treated using depth filters with two or more different pore sizes. The pore size of the depth filters 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.

[0026] When depth filters with 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 filters. The turbidity may be measured by a known method, and the turbidity of the disruption solution after passing through the depth filters 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 then 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 below, preferably 15°C or below, more preferably 10°C or below, and even more preferably 5°C or below.

[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 difference in elevation 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. Ultrafiltration may be performed using two or more types of ultrafiltration membranes. Commercially available ultrafiltration membranes, such as the TFF Omega membrane (manufactured by Cytiva), may be used. 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 pretreatment depth filter 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), SC050B020 (pore size: 0.4 μm to 1 μm), SC050PDD1 (pore size: 0.1 μm to 0.85 μm), SC050PDE2 (pore size: 0.1 μm to 0.85 μm), 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 called "diafiltration" which performs filtration by passive diffusion. Alternatively, ultrafiltration may be a method in which a solution is introduced into an ultrafiltration membrane at a constant flow rate. When ultrafiltration is performed, the disruption solution after passing through the depth filter may be directly filtered. The mixing ratio of the disruption solution to a solvent such as water is not particularly limited as long as ultrafiltration can be performed.

[0034] The ultrafiltration temperature 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 liquid volume is reduced by at least 2 times, preferably at least 5 times, more preferably at least 10 times.

[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 a depth filter 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] Static ultrafiltration treatment The method of this embodiment may include a step of allowing the disruption liquid derived from plants, mushrooms, or microalgae to stand and separate into a water layer and a contaminant layer.

[0041] The lysate 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 in the depth filter-ultrafiltration process above.

[0043] Before the step of allowing the disruption solution to stand, the method may include a step of passing the disruption solution through a filter mesh. 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 at a very low temperature or a low temperature (approximately -200°C to -30°C). Freeze-drying, also known as vacuum freeze-drying, 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, preferably an anion-exchange membrane. [Example]

[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 the centrifugation method Parsley was washed 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 JE (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 EV samples.

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

[0056] (1-3) Preparation of EVs using the static method Parsley was washed in a plastic bucket, drained, and then juiced using a household juicer (HUROM). The juice was passed through a 132 μm filter mesh and left overnight at 4°C. The next day, the sedimentation of larger fragments was confirmed. The supernatant was then pretreated using a membrane depth filter (PDP8, PDE2, Cytiva) similar to that used for centrifugation, followed by ultrafiltration using a TFF Omega membrane (Cytiva, nominal molecular weight cutoff: 100 kDa or 500 kDa) to obtain EV samples.

[0057] (1-4) Nano Tracking Analysis (NTA) The particle size, size distribution, and concentration of extracellular vesicles obtained by the centrifugation, depth filtration, and static methods 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 the centrifugation and depth filtration methods, and Figure 2A shows the results for EV samples prepared by the centrifugation and static methods. The results in Figures 1 and 2 show that EVs prepared by the centrifugation method contained an average size of 156 nm (50-600 nm). Furthermore, parsley EVs prepared by the depth method contained an average size of 111 nm (50-250 nm), and those prepared by the static method contained 153 nm (50-600 nm). These results demonstrate that the depth method can prepare EVs with a more specific size range 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: InvivoGen) were cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 μg / ml Normocin (InvivoGen Scientific), and Pen-Strep (100 U / ml) at 37°C in a 5% CO2 incubator. 6 The cells were subcultured at a rate of 100mm cells / 100mm dish.

[0059] (1-6) LPS stimulation treatment 0.8x10 Raw-Blue cells 6 2.5x10 cells / well were seeded in a 12-well dish, and EVs were added at 2.5x10 9 and 1.0x10 10After incubation at 1000pF for 16 hours at 1000pF / ml, 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 them using TRIsure (manufactured by Nippon Genetics).

[0060] (1-7) Evaluation of the effect of EVs on the expression of inflammatory cytokines in mouse macrophage cells after LPS stimulation 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 these cytokines as an indicator. IL-6 and IL-1β expression levels were measured by RT-qPCR using RNA extracted from LPS-stimulated mouse macrophage cells. Gapdh was used as a correction gene, and the expression level in LPS-stimulated cells was set to 1 in the presence of PBS instead of EVs. The values ​​are shown as relative values ​​(fold change). We used RNA-seq analysis to evaluate the effect of EVs on the expression of inflammatory cytokines in mouse macrophage cells stimulated with LPS. RNA extracted from LPS-stimulated mouse macrophage cells was used to prepare an mRNA-seq library using polyA selection and sequenced using NavaSeq (Illumina). Sequencing yielded 6 Gb of data (approximately 20 million reads) using a 150 bp paired-end method. A gene expression matrix was generated using 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 identified as significantly altered genes. The expression levels of each gene for each sample were normalized 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 treated with EVs prepared by the centrifugation method and the depth method and stimulated with LPS. Figure 2B shows the expression levels of IL-6 and IL-1β when mouse macrophage cells were treated with 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 the 1,042 genes. Parsley EVs prepared by the depth method significantly suppressed the expression of inflammatory cytokines such as CXCL2, CXCL3, and TGFB1 in addition to IL-6 and IL-1B.

[0063] The results of Figures 1 and 2 show 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 effects than parsley EVs prepared by centrifugation.

Claims

1. Passing the crushed solution of plants, mushrooms, or microalgae through a depth filter; and A step of ultrafiltration of the disrupted solution after passing through the depth filter. A method for producing extracellular vesicles, comprising: The method wherein the depth filter is comprised 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. 3. The method of claim 2, wherein the at least two depth filters comprise depth filters with pore sizes of 10 to 300 μm and 0.5 to 75 μm.

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

Images