Method for producing exosome-containing liquid derived from mesenchymal stem cells and pharmaceuticals containing exosome-containing liquid

Abstract

To provide a method for easily and mass-producing a highly concentrated exosome-containing liquid [Solution] The present inventors have discovered that a highly concentrated exosome-containing solution can be produced in large quantities and easily by disrupting mesenchymal stem cells. Specifically, the method comprises: (1) disrupting mesenchymal stem cells; (2) stirring the disrupted solution obtained in step (1) using a stirrer or a vortex mixer; and (3) filtering the disrupted solution after stirring. The exosome-containing solution produced by the present invention is cell-free, making it easy to prepare and handle, and can be stored for long periods by freezing. Furthermore, because no additives are added, there is no need to worry about additives affecting the exosomes, providing significant advantages in research and clinical applications.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a liquid containing exosomes derived from mesenchymal stem cells and a pharmaceutical product containing the liquid containing exosomes. [Background technology]

[0002] Mesenchymal stem cells have the ability to self-proliferate and differentiate into various cells, including chondrocytes, osteocytes, muscle cells, and adipocytes. Recently, attention has been focused on the various factors secreted by mesenchymal stem cells (paracrine factors) rather than the differentiation ability of the mesenchymal stem cells themselves. It has been found that mesenchymal stem cells secrete cytokines, chemokines, growth factors, and exosomes that have anti-inflammatory, pro-angiogenic, and pro-cell proliferation effects, and promote tissue repair through the paracrine effect (see, for example, Non-Patent Document 1).

[0003] Exosomes (also called "exosomes") are vesicles composed of a lipid bilayer and are secreted extracellularly by cells (cells of origin). Exosomes encapsulate various proteins, lipids, mRNAs, microRNAs, and cell-specific components that reflect the biological functions of the cell of origin. Because exosomes can fuse with distant cells and release their contents, they are believed to play a functional role in mediating cell-cell communication and cellular immunity. Therefore, in recent years, the therapeutic effects of exosomes on various diseases have been studied. For example, their use as a drug delivery system for delivering various therapeutic agents to target cells has been investigated (see, for example, Non-Patent Documents 2 and 3), and their use as a biomarker for neurological diseases such as Alzheimer's disease has also been studied (see, for example, Non-Patent Document 4).

[0004] The properties of exosomes vary depending on the cell type of origin. In particular, exosomes derived from mesenchymal stem cells are known to exhibit the therapeutic effects of stem cells in regenerative medicine, and research into the use of exosomes in wound and fracture healing and regenerative medicine is actively being conducted.

[0005] For clinical and research use of exosomes, it is necessary to produce large quantities of high-quality exosomes. Methods for mass-producing exosomes include a method using a "stem cell-derived exosome production-promoting composition" that increases the number of stem cell-derived exosomes by culturing stem cells in a cell culture medium containing a specific compound (see, for example, Patent Document 1), and a method for producing exosomes by culturing stem cells in a cell population containing mesenchymal stem cell precursors in which 80% or more of the cells express CD73 + Examples of such methods include a method using a cell population containing a specific marker protein such as the above (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2022-533277 [Patent Document 2] Special Publication No. 2022-542158 [Non-patent literature]

[0007] [Non-Patent Document 1] Chamberlain G., Fox J., Ashton B., Middleton J., “Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.” Stem Cells. 25 (2007) 2739-2749 [Non-patent document 2] Emmanouilidou E. et al., “Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival”, J Neurosci. 2010 May 19;30 (20) : 6838-51 [Non-patent document 3] Ian Fyfe, “Exosomes can spread toxic AD pathology” Nat Rev Neurol. 2018 Aug;14(8) : 451 [Non-patent document 4] Yamayoshi A., et al., “Development of Novel Drug Delivery System Targeting Exosomal microRNA” The Pharmaceutical Society of Japan, Vol. 140, No.5, 625-631 (2020) Summary of the Invention [Problem to be solved by the invention]

[0008] The quality of exosomes is affected by the type of cells from which they are derived and the conditions under which they are produced. Therefore, it is preferable not to add additives or reagents. However, the technology described in Patent Document 1 produces exosomes by adding compounds not found in living organisms, such as exendin-4, and it is unclear to what extent this adversely affects the cells and exosomes. In addition, a washing step is required to wash away the additives, which is a laborious process. Furthermore, the technology described in Patent Document 2 requires progenitor cells of mesenchymal stem cells, and the method for producing these progenitor cells is time-consuming and labor-intensive.

[0009] Therefore, the present invention provides a method for easily and mass-producing a highly concentrated exosome-containing liquid without adding artificial compounds such as exosome production promoters. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors focused on mesenchymal stem cells and discovered that a highly concentrated exosome-containing solution can be produced in a large amount and easily by disrupting the cells, thereby completing the present invention.

[0011] That is, the present invention has the following configuration. [1] A method for producing a liquid containing exosomes derived from mesenchymal stem cells, comprising the following steps: (1) a step of disrupting mesenchymal stem cells; (2) Stirring the disruption solution obtained in step (1) using a stirrer or a vortex mixer; (3) A step of filtering the disruption liquid after the stirring treatment. [2] The method for producing an exosome-containing liquid according to [1], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. [3] The method for producing an exosome-containing fluid according to [1] or [2], wherein the step (1) is carried out by freezing and thawing or ultrasonication. [4] In the step (2), The method for producing an exosome-containing solution according to [1] or [2], wherein the disruption solution is stirred using a vortex mixer at 100 to 3,500 rpm for 1 to 30 minutes. [5] A pharmaceutical comprising, as an active ingredient, an exosome-containing liquid produced using the method described in [1] or [2]. [6][5] The medicines listed in [6][5] are administered intravenously Used to be administered , promotes fatigue recovery Agent . [7] A supplement for non-human mammals, comprising, as an active ingredient, an exosome-containing liquid produced using the method described in [1] or [2]. [Effects of the Invention]

[0012] The production method of the present invention enables the easy and large-scale production of highly concentrated exosome-containing solutions. Since expensive machinery such as ultracentrifuges is not required, production costs can be reduced. Furthermore, because cell fragments, rather than live cells, are used for production, the resulting exosome-containing solutions are cell-free. Therefore, the exosome-containing solutions of the present invention pose little risk of cell-derived infection or carcinogenesis, and there is no risk of microvascular blockage when administered as pharmaceuticals. Unlike live cells, there is no need for cell culture at the appropriate time for use. Preparation and handling are easy, and long-term storage by freezing is also possible. Furthermore, the present invention does not require the addition of additives such as exosome production promoters. Since there is no need to worry about the effects of additives on exosomes, this method offers significant advantages in research and clinical applications. Furthermore, pharmaceuticals and supplements containing the exosome-containing solutions of the present invention as active ingredients are expected to promote fatigue recovery. [Brief explanation of the drawings]

[0013] [Figure 1] Measurement results of exosome amount in exosome-containing liquid DETAILED DESCRIPTION OF THE INVENTION

[0014] <Exosomes> In the present invention, an "exosome" refers to an endoplasmic reticulum composed of a lipid bilayer that is secreted from a cell to the outside, and the lipid bilayer contains proteins, lipids, nucleic acids (miRNA, mRNA, DNA, etc.), and the like.

[0015] Exosomes are endoplasmic reticulum that are secreted extracellularly from cells. Exosome formation begins with the formation of early endosomes by endocytosis, which engulf receptors present on the plasma membrane. The early endosomes transition to late endosomes, which then invaginate to form intraluminal membrane vesicles (ILVs). Endosomes containing many ILVs (multivesicular bodies: MVBs) eventually fuse with the plasma membrane and are released from the cell.

[0016] The present inventors focused on the mesenchymal stem cells that secrete exosomes, and succeeded in extracting exosomes contained within the cells before secretion in addition to the secreted exosomes by disrupting the cells.

[0017] <Mesenchymal stem cells>

[0018] In the present invention, the mesenchymal stem cells from which exosomes are secreted are cells belonging to the mesenchymal system, and are known to have pluripotency and self-renewal capabilities, as well as the ability to differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes, as well as into nerve cells and cardiomyocytes.

[0019] Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. However, adipose tissue-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, and among these, adipose tissue-derived mesenchymal stem cells are preferred because they can be easily collected.

[0020] Adipose tissue-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are prepared according to conventional methods. Here, a method for preparing adipose tissue-derived mesenchymal stem cells, which are preferred cells, is specifically described.

[0021] The biological origin of the mesenchymal stem cells of the present invention is not particularly limited, and includes humans as well as non-human mammals (including pet animals, livestock, and laboratory animals, specifically monkeys, pigs, cows, horses, goats, sheep, dogs, cats, camels, mice, rats, guinea pigs, and hamsters). When administering the exosome-containing liquid of the present invention as a pharmaceutical, it is preferable to collect mesenchymal cells from the same individual as the recipient (autologous). However, this does not preclude the use of mesenchymal stem cells from the same species of animal (allogeneic) or mesenchymal stem cells from a different species of animal. <Adipose tissue-derived mesenchymal stem cells>

[0022] Adipose tissue-derived mesenchymal stem cells (also known as Adipose-derived stem cells (ADSCs (ASCs), Adipose-derived regeneration cells (ADRCs), Adipose tissue-derived mesenchymal stem cells (AT-MSCs), AD-MSCs, etc.; hereafter simply referred to as "ADSCs")) are a type of somatic stem cell found in adipose tissue. Adipose tissue-derived mesenchymal stem cells also possess self-renewal and multipotency, and are known to be capable of differentiating into a variety of cells, including not only fat cells but also bone, cartilage, nerves, muscles, cardiac muscle, blood vessels, hepatocytes, and pancreatic islet cells. <Method for preparing adipose tissue-derived mesenchymal stem cells>

[0023] In the present invention, ADSCs also include cells obtained by culturing (including subculture) somatic stem cells, as long as they maintain pluripotency. ADSCs are typically prepared in an "isolated state" using adipose tissue isolated from a living body as the starting material, as cells that constitute a cell population (including cells other than ADSCs derived from adipose tissue). Here, "isolated state" refers to a state in which the cells have been removed from their original environment (i.e., the state in which they constituted part of a living body), i.e., they exist in a state different from their original state due to artificial manipulation.

[0024] In the present invention, ADSCs can be prepared according to standard methods. Because ADSCs are widely used for a variety of purposes, those skilled in the art can prepare them by referring to literature and textbooks. Cells provided from public cell banks or commercially available cells may also be used. Below, as an example of a cell preparation method, a method for preparing adipose tissue-derived mesenchymal stem cells (one example) will be described.

[0025] ADSCs are prepared through steps such as isolating, washing, concentrating, and culturing stem cells from adipose tissue. The method for preparing ADSCs is not particularly limited. For example, ADSCs can be prepared according to known methods (see, for example, Fraser JK et al. (2006), Fat tissue: an under-appreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24(4): 150-4. Epub 2006 Feb 20. Review; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec; 13(12): 4279-95; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7(2): 211-28). Additionally, devices for preparing ADSCs from adipose tissue (e.g., the Celution® device (Cytori Therapeutics, Inc., San Diego, USA)) are commercially available, and ADSCs can be prepared using such devices. Using such devices, a cell population containing ADSCs can be isolated from adipose tissue (K. Lin. Et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). A specific example of a method for preparing ADSCs is described below. (1) Preparation of cell populations from adipose tissue

[0026] Adipose tissue is collected from humans and non-human mammals by means of excision, suction, or other methods. Non-human mammals can be any of the animals mentioned above, including pets, livestock, and laboratory animals. There are no particular restrictions on the age or sex of the organism. In humans, ADSCs can also be prepared from tissue fragments aspirated during liposuction procedures for cosmetic surgery, or from excised adipose tissue contained in tissue removed from the body during surgery. Because ADSCs reside around large blood vessels, they can be obtained in greater quantities from excised adipose tissue than from lipoaspirate. Meanwhile, preparing stem cells from lipoaspirate leaves smaller surgical scars and places less strain on the donor.

[0027] Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat is a preferred cell source because it can be collected very easily under local anesthesia, placing less strain on the donor during collection. While one type of adipose tissue is usually used, two or more types of adipose tissue can also be used in combination. Additionally, adipose tissue collected in multiple batches (which do not have to be the same type) can be mixed and used for subsequent procedures.

[0028] The amount of adipose tissue to be collected can be determined taking into consideration the type of donor, the type of tissue, or the amount of ADSC required, and is, for example, approximately 0.5 to 500 g. However, taking into consideration the burden on the donor, it is preferable to collect an amount of approximately 3 to 20 g or less at one time. The collected adipose tissue is optionally subjected to removal of blood components adhering to it and to fragmentation, and then subjected to the following enzymatic treatment. Blood components can be removed by washing the adipose tissue in an appropriate buffer solution or culture medium.

[0029] The enzyme treatment is carried out by digesting the adipose tissue with an enzyme such as collagenase, trypsin, or dispase. Such enzyme treatment may be carried out by techniques and conditions known to those skilled in the art (see, for example, RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). The cell population obtained by the above enzyme treatment contains pluripotent stem cells, endothelial cells, stromal cells, blood cells, and / or their progenitor cells. The types and ratios of cells constituting the cell population depend on the origin and type of adipose tissue used. (2) Obtaining the sedimented cell population (SVF fraction: stromal vascular fractions)

[0030] The cell population is then centrifuged. The sediment from the centrifugation is collected as a sedimented cell population (also referred to herein as the "SVF fraction"). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes at 800 to 1,500 rpm. Prior to centrifugation, the cell population after enzyme treatment is preferably filtered to remove any undigested tissues present. The "SVF fraction" obtained here contains ADSCs. The type and ratio of cells constituting the SVF fraction depend on the origin and type of adipose tissue used, the conditions of enzyme treatment, and other factors. The characteristics of the SVF fraction are described in International Publication No. 2006 / 006692A1. (3) Selective culture of adherent cells (ADSCs) and cell recovery

[0031] The SVF fraction contains not only ADSCs but also other cellular components (endothelial cells, stromal cells, blood cells, their progenitor cells, etc.). Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cellular components from the SVF fraction. The cells obtained as a result are then used as ADSCs in the present invention. First, the SVF fraction is suspended in an appropriate medium, then seeded on a culture dish and cultured overnight. Floating cells (non-adherent cells) are removed by changing the medium. Thereafter, the culture is continued while changing the medium appropriately (for example, once every 2 to 4 days). Subculture is performed as necessary. The number of passages is not particularly limited, but excessive repeating of passages is not preferable from the perspective of maintaining pluripotency and proliferation ability (it is preferable to limit it to about 6 passages). Note that a medium for ordinary animal cell culture can be used as the culture medium. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. may be used. Media supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or serum substitutes (e.g., knockout serum replacement (KSR)) may also be used. The amount of serum or serum substitute added can be set within the range of, for example, 5 to 30% (v / v).

[0032] The above procedure selectively allows adherent cells to survive and proliferate. The proliferated cells are then harvested. The harvesting procedure can be performed using standard methods. For example, cells can be easily harvested by treating them with enzymes (trypsin or dispase) and then detaching them with a cell scraper or pipette. Furthermore, when sheet culture is performed using commercially available temperature-sensitive culture dishes, it is possible to harvest the cells directly in sheet form without enzyme treatment. Using the cells (ADSCs) harvested in this way, a cell population containing highly purified ADSCs can be prepared. (4) Low-serum culture (selective culture in low-serum medium) and cell recovery

[0033] In one embodiment of the present invention, the following low-serum culture is performed instead of or after the above procedure (3). The resulting cells are then used as ADSCs in the present invention. In low-serum culture, the SVF fraction (if this step is performed after (3), the cells recovered in (3) are used) is cultured under low-serum conditions to selectively proliferate the target pluripotent stem cells (i.e., ADSCs). Because low-serum culture requires only a small amount of serum, when using ADSCs obtained by the method of the present invention for therapeutic purposes, it is possible to use the subject's (patient's) own serum. In other words, culture using autologous serum is possible. Here, "low-serum conditions" refers to conditions in which the medium contains 5% (v / v) or less serum. Preferably, the cells are cultured in a culture medium containing 2% (v / v) or less serum. More preferably, the cells are cultured in a culture medium containing 2% (v / v) or less serum and 1 to 100 ng / mL fibroblast growth factor-2 (bFGF). Serum-free medium may also be used.

[0034] The serum is not limited to fetal bovine serum; human serum, sheep serum, etc. can also be used. When the activated sperm obtained by the method of the present invention are used for human treatment, human serum is preferably used, and serum from the subject of treatment (i.e., autologous serum) is more preferably used. Conventional animal cell culture media can be used, provided that the serum content is low at the time of use. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used.

[0035] By culturing using the above method, ADSCs can be selectively proliferated. Furthermore, because ADSCs proliferate under the above culture conditions have high proliferation activity, the number of cells required for the present invention can be easily prepared by subculture. International Publication No. 2006 / 006692A1 describes the characteristics of cells that selectively proliferate when the SVF fraction is cultured in low serum. Subsequently, the cells selectively proliferated by the above low serum culture are recovered. The recovery procedure can be performed in the same manner as in (3) above. By using the recovered ADSCs, a cell population containing highly purified ADSCs can be obtained.

[0036] In the above-described methods, cells proliferated by low-serum culture of the SVF fraction are used; however, cells proliferated by low-serum culture of a cell population obtained from adipose tissue directly (without centrifugal treatment to obtain the SVF fraction) may also be used as ADSCs. That is, in one aspect of the present invention, cells proliferated when a cell population obtained from adipose tissue is cultured in low serum are used as ADSCs. Furthermore, the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as is, rather than the pluripotent stem cells obtained by selective culture (above (3) and (4)). Note that "used as is" here means used in the present invention without undergoing selective culture. <Method for preparing exosome-containing liquid> (Step (1): Disrupting ADSCs)

[0037] Common cell disruption methods can be used to disrupt ADSCs. Examples include freeze-thawing (a process of freezing and then thawing), ultrasound, a French press, a mortar, a homogenizer, and glass beads. Furthermore, cells subjected to disruption are not limited to live cells; dead or damaged cells can also be used. Among the above disruption methods, freeze-thawing and ultrasound are preferred. Freeze-thawing is particularly preferred because it is simple and convenient, and it is hygienic, avoiding contamination due to contact between the machine and the cells. When disrupting by freeze-thawing, the preferred conditions described below can be used. When disrupting by ultrasound, it is preferable to use unfrozen cells. However, because heat generated by the equipment often causes protein denaturation and aggregation, it is preferable to repeatedly perform short-term treatments while cooling the cell suspension on ice. Specifically, it is preferable to disrupt cells for 5 to 15 seconds at an output of 200 to 300 W, followed by a 10 to 30 second break, repeated multiple times.

[0038] During the freezing process, cells expand and form ice crystals, which then destroy the cells and dissolve upon thawing. Therefore, repeated freeze-thawing is preferable to achieve sufficient dissolution. Specifically, the freeze-thaw process is preferably repeated 1 to 5 times. Repeating 2 to 4 times is even more preferable. Examples of freezing methods include using a freezer, deep freezer, liquid nitrogen, etc. The freezing temperature in the freeze-thaw process is not particularly limited as long as it is a temperature at which the entire cell population can be frozen, and is preferably -20°C to -196°C. More specifically, the upper limit is preferably -20°C or lower, more preferably -30°C or lower, and even more preferably -70°C or lower.

[0039] The cooling time is not particularly limited as long as the entire cell population can be frozen, but when using a freezer or refrigerator, it is preferable to leave it in the refrigerator for at least 1 hour, preferably at least 10 hours, to allow sufficient cooling. The upper limit of the cooling time is not particularly limited, but is preferably within 72 hours, more preferably within 48 hours, and even more preferably within 12 hours. It is efficient to place the cells in a refrigerator or freezer before returning home, cool them overnight, and then work on them the next day. When using liquid nitrogen, the container containing the cells can be cooled directly or by placing the cells in a cryopreservation container and immersing the container in liquid nitrogen.

[0040] Frozen cells can be thawed by any method. For example, frozen cells can be placed in a water bath, incubator, refrigerator, or thermostatic bath at a temperature higher than the freezing point. Specifically, thawing can be performed by leaving the cells in a refrigerator at 5°C or below overnight, by thawing in a water bath (e.g., 35-40°C), or by thawing at room temperature. The cell suspension concentration used for disruption is 1 x 10 ADSCs. 4 ~1×10 8 cells / mL is preferred. ADSCs are 10 × 10 4 ~1000×10 4 cells / mL is more preferred, and 10 x 10 4 ~500×10 4 cells / mL is more preferable. This is an easy-to-work-with concentration, and a sufficient amount of exosome-containing solution can be obtained in one operation. (Step (2): Stirring the disruption solution)

[0041] The lysate is placed in a container such as a test tube and stirred using a stirrer or vortex mixer. Using a stirrer requires subsequent cleaning of the stirrer, and there is also the risk of exosome loss due to adhesion of the lysate to the stirrer. Therefore, stirring using a vortex mixer is preferred to improve production efficiency. To obtain a highly concentrated exosome-containing solution, the rotation speed of the vortex mixer is preferably 100 rpm to 3,500 rpm, more preferably 1,500 rpm to 2,700 rpm. The stirring time is preferably 1 minute to 30 minutes, more preferably 3 minutes to 25 minutes. To efficiently obtain a highly concentrated exosome, a short stirring time is preferred, with 1 second to 2 minutes being preferred.

[0042] It is believed that stirring the bottom of the container by rotating it at high speed with a vortex mixer can efficiently extract exosomes from the disrupted cells, leading to the production of a highly concentrated exosome-containing solution. Note that the temperature during the stirring process is not particularly limited.

[0043] Alternatively, the disrupted solution after stirring may be centrifuged, and the resulting supernatant may be used in the next step (filtering). Centrifuging the disrupted solution after stirring removes nuclei and other particles from the supernatant, preventing clogging of the filter and allowing for efficient filtering. When centrifuging, the solution is preferably centrifuged at 100 to 1,500 × g for 3 to 10 minutes. The temperature during centrifugation is not particularly limited. (Step (3): Filtering the disruption solution after stirring or the supernatant after centrifugation)

[0044] The disrupted solution after stirring or the supernatant obtained by centrifugation is filtered. Filter filtration can remove unwanted components. Furthermore, by using a filter with an appropriate pore size, removal of unwanted components and sterile filtration can be performed simultaneously. The material of the filter used for filtration is not particularly limited, but cellulose acetate or metal filters, which are less likely to adsorb proteins, are preferred. Cellulose acetate is particularly preferred. The filter pore size is preferably 0.1 to 0.45 μm, more preferably 0.15 to 0.3 μm. If sterile filtration is also performed simultaneously, a pore size of 0.2 μm is preferred. Note that "filter filtration" in the present invention is a physical separation method that uses gravity to remove unwanted components. It differs in both its principle and the components separated from centrifugation, which uses centrifugal force to separate precipitates and supernatants by utilizing factors such as centrifugal acceleration, viscosity, and density differences between particles and fluids.

[0045] If the filtrate is not to be used immediately for treatment, it can be frozen and stored until use. It is preferably stored at -100 to -60°C. Generally, repeated freezing and thawing of cells tends to reduce cell activity and increase the number of dead cells. However, because the exosome-containing liquid of the present invention does not contain stem cells, its quality remains unchanged no matter how many times it is frozen and thawed.

[0046] The exosome-containing liquid of the present invention can be used as a raw material without separating exosomes, or exosomes can be separated and used. There are no limitations on the method for separating exosomes from the exosome-containing liquid of the present invention, and exosomes can be separated using methods such as filter filtration, gel filtration chromatography, electrophoresis, and combinations thereof. Although this equipment is expensive, exosomes can also be separated from the exosome-containing liquid using an ultracentrifuge.

[0047] Confirmation that the exosomes obtained according to the present invention are the intended ones can be carried out by morphological observation using a transmission electron microscope, genetic analysis using PCR, immunological techniques (ELISA, FACS, etc.), Western blotting, etc. <Exosome-containing liquid of the present invention>

[0048] The exosome-containing liquid of the present invention contains 1×10 6 When CD63 protein levels were measured using a cell suspension of 1000 cells / mL using the CD9xCD63 ELISA Kit for Quantitating Human Exosomes (Cosmo Bio Co., Ltd.), the exosome content was preferably 10 to 10,000 pg / mL, more preferably 50 to 7,500 pg / mL, more preferably 75 to 5,000 pg / mL, and even more preferably 100 to 3,000 pg / mL.

[0049] According to the production method of the present invention, not only exosomes secreted outside cells but also exosomes contained within cells can be efficiently extracted. Therefore, a highly concentrated exosome-containing solution can be produced. Furthermore, since the method does not require expensive machinery such as an ultracentrifuge and the operation is very simple, production costs can be reduced.

[0050] Furthermore, because the exosome-containing liquid of the present invention does not contain live cells, it is less susceptible to the risks of live cell-induced carcinogenesis, etc., and is not only free from the problem of transplant rejection, but also free from the risk of causing microvascular blockage when administered as a pharmaceutical. It is easy to prepare and handle, and can be stored for long periods by freezing. Furthermore, the exosome-containing liquid of the present invention not only contains a large amount of exosomes, but also contains large amounts of various growth factors that were contained within cells. <Uses of the exosome-containing liquid of the present invention>

[0051] The exosome-containing liquid obtained by the production method of the present invention can be used for various purposes, such as pharmaceuticals, supplements, cosmetics, and foods, as described below. It can be used as an exosome-containing liquid, or exosomes can be separated from the liquid and used. Furthermore, it can be used in the form of a composition by mixing the exosome-containing liquid with an excipient, etc.

[0052] For example, when used as a pharmaceutical, the pharmaceutical may contain, as an active ingredient, the exosome-containing liquid or isolated exosomes obtained by the production method of the present invention. Exosomes are packed with intracellular components such as DNA, mRNA, miRNA, and proteins, and are said to have various physiological functions. In particular, the exosome-containing liquid of the present invention not only contains a large amount of exosomes, but is also thought to contain a large amount of various growth factors, making it effective for tissue regeneration.

[0053] Specifically, it can be used as a therapeutic agent for central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and sequelae of cerebral infarction; respiratory diseases such as interstitial lung disease and chronic obstructive pulmonary disease; heart diseases such as myocardial infarction and heart failure; kidney diseases such as nephritis (including chronic nephritis) and nephrotic syndrome; pancreatic diseases such as diabetes and pancreatitis; and inflammatory bowel diseases such as inflammatory bowel disease and Crohn's disease. It can also be used as a therapeutic agent for diseases that cause orthopedic / motor dysfunction, such as rheumatism, osteoarthritis of the knee, hernias, and diseases that cause difficulty in maintaining balance, as well as diseases that cause muscle weakness, such as amyotrophic lateral sclerosis. The exosome-containing liquid of the present invention is also believed to have the following effects: significantly enriching cumulus cells, significantly reducing reactive oxygen species (ROS), improving mitochondrial distribution and activity, reducing early apoptosis in oocytes, improving nuclear maturation and in vitro fertilization, improving the developmental potential of in vitro fertilized eggs, and improving the quality of blastocyst-stage embryos. It is also said to improve sperm motility, and therefore can be used for the treatment and improvement of infertility caused by decreased egg or sperm function, oligospermia, endometriosis, etc., improving the success rate of in vitro fertilization, and for livestock breeding and species maintenance (e.g., preserving endangered species, maintaining or crossbreeding pet lines), etc. Furthermore, due to their lipid bilayer structure, exosomes are said to be able to penetrate into the dermis layer of the skin, where they are said to have skin regeneration and anti-aging effects through the proliferation and activation of fibroblasts, which are cells that make up the skin, as well as promoting collagen synthesis, and therefore can also be used for anti-aging and cosmetic purposes.

[0054] Alternatively, exosomes can be incorporated into various tissues and are expected to have a restorative effect on tissues with impaired function, making them effective for promoting fatigue recovery. Fatigue causes a decrease in activity efficiency. Furthermore, if fatigue accumulates chronically, it can become a factor in inducing disease. Therefore, the pharmaceutical product of the present invention is also effective as a preventive drug to prevent the onset of disease.

[0055] Alternatively, the exosome-containing liquid of the present invention is preferably used as a supplement for non-human mammals, containing the exosome-containing liquid as an active ingredient. Here, a supplement is a concentrated source of nutrients or other substances with nutritional or physiological effects intended to supplement a normal diet, and has the function of helping to maintain health or recover from fatigue.

[0056] Pharmaceuticals containing the exosome-containing liquid of the present invention as an active ingredient may contain non-toxic, inert, pharmaceutically acceptable excipients, such as solid, semi-solid, or liquid diluents, dispersants, fillers, and carriers. Furthermore, within the scope that does not impair the effects of the present invention, stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, flow enhancers, flavoring agents, colorants, flavoring preservatives, antibacterial agents, vehicles, physiological saline, antibacterial agents, and other medicinal agents may be contained as additives.

[0057] In particular, pharmaceuticals containing the exosome-containing liquid of the present invention as an active ingredient preferably contain an antibacterial agent. Antibacterial agents refer to compounds with antibacterial activity, such as antibiotic-antimycotic solution, isopropylmethylphenol, ginger oil, thymol, chlorhexidine hydrochloride, and cetylpyridinium chloride. These antibacterial agents are preferably contained in an amount effective to exert their antibacterial effect. By including an antibacterial agent in the pharmaceutical of the present invention, the growth of infectious bacteria is suppressed, ensuring safety for a certain storage period.

[0058] The dosage form of the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient is not particularly limited, and it may be liquid or solid, or may be processed into a powder form by freezing and / or drying, and may be prepared in various dosage forms depending on the intended use, such as powder, granules, tablets, capsules, injections, infusions, inhalants, ointments, eye drops, nasal drops, and transdermal absorption formulations.

[0059] The method of administration of a pharmaceutical comprising the exosome-containing liquid of the present invention as an active ingredient is not particularly limited, and may be local administration or systemic administration. Examples include subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, arterial injection, intraperitoneal injection, intrathoracic injection, direct injection to a local area, and direct application. In particular, intravenous injection allows the drug to be immediately transported throughout the body via the bloodstream, resulting in a high blood concentration. Therefore, when an immediate effect is required, it is preferable to administer a pharmaceutical comprising the exosome-containing liquid of the present invention as an active ingredient by intravenous injection.

[0060] The dosage of the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient may be an effective amount as long as it provides a therapeutic effect. The dosage level depends on the individual's type, severity, age, sex, type of disease, etc. The administration frequency is such that the pharmaceutical of the present invention provides a therapeutic effect against the disease. The administration period is such that the pharmaceutical of the present invention provides a therapeutic effect against the disease.

[0061] The subjects of the present invention include humans and non-human mammals for which treatment is desired or required, such as monkeys, pigs, cows, horses, goats, sheep, dogs, cats, camels, mice, rats, guinea pigs, and hamsters, as well as pet animals, livestock, and laboratory animals. [Example]

[0062] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way. (1) Preparation of exosome-containing solution <Preparation 1 of ADSC Disrupted Filtrate (Freezing and Thawing)>

[0063] Using human adipose-derived stem cells (human A), the filtrate was prepared by the method described in this specification. Specifically, the human adipose-derived stem cells were cultured in MesenPro RS medium to proliferate ADSCs. The cells were subcultured 6 times while changing the medium every 2 days until 5×10 6 cells were obtained. The concentration was adjusted using PBS to prepare a cell suspension of 1×10 6 cells / mL. They were frozen in a -20°C freezer for 1 day and then in an -80°C deep freezer for 12 hours. Although liquid nitrogen (-196°C) can freeze instantly, here the container containing the cell suspension was immersed in liquid nitrogen for 30 minutes until the start of measurement.

[0064] After that, the frozen cell solution was placed in a 4°C refrigerator and thawed until thawing was confirmed by visual inspection. After disrupting the cells in this way, stirring treatment was performed using a vortex mixer for the stirring time described in Table 1. The rotation speed was 2,500 rpm. Next, the liquid after the stirring treatment was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain an exosome-containing solution. As a control, a serum-free medium (KBM ADSC-5, Cosmo Bio Co., Ltd.) was used. <Quantitative Analysis of Exosomes by ELISA>

[0065] The amount of exosomes in the exosome-containing solution, culture supernatant, and serum-free medium prepared above was measured as the protein amount of CD63 using a human-derived exosome quantification CD9×CD63 ELISA kit manufactured by Cosmo Bio Co., Ltd. The measurement method followed the protocol of the kit manufacturer. The results are shown in Table 1 and Figure 1. Figure 1 is a graph of the results in Table 1.

[0066] As is clear from Table 1 and Figure 1, the exosome-containing solution obtained by vortexing and filter-filtering the freeze-thawed sample contained a high concentration of exosomes. The lower the freezing temperature, the more remarkable the effect. In particular, compared with the control (culture supernatant), Example 3-3 contained about 30 times as many exosomes.

[0067] Thus, according to the production method of the present invention, a high-concentration exosome-containing solution can be produced in large quantities and simply. The use of exosomes for various purposes is expected.

Table 1

[0068] Using human (human B) adipose stem cells, they were cultured in MesenPro RS medium to proliferate ADSC. The cells were subcultured 6 times while changing the medium every 2 days until 5×10 6 cells were obtained. The concentration was adjusted using PBS to prepare a cell suspension of 1×10 6 cells / mL. Thereafter, while changing the operating conditions (freezing conditions, number of freeze-thaws, vortex time), the same operations as in <Adjustment 1 of ADSC Disruption Filtrate (Freeze-Thaw)> were performed. The operating conditions and results are shown in Table 2. When the stirring time was 1 minute or less, a higher-concentration exosome-containing solution was obtained. Short-time stirring operations are preferable because of their excellent operability.

Table 2

[0069] (2) Evaluation of Exosome-Containing Solution in Dogs (Effect on Chronic Nephritis) Using canine mesenchymal stem cells (beagle, 3-year-old male), an exosome-containing solution was prepared in the same manner as for human adipose stem cells. In addition, canine cells that satisfied the following requirements were used. · Having received mixed vaccination, filaria prevention, and rabies prevention vaccination every year, · Having not received a blood transfusion in the past, - Absence of systemic infectious skin diseases, - Not having previously contracted or suspected of contracting a blood-borne infectious disease (such as babesiosis or canine brucellosis), -No serious metabolic or endocrine disorders -Not suffering from any genetic diseases, - Not suffering from malignant tumors, Canine ADSCs that met the above requirements were collected, expanded, and cultured for 6 passages, yielding 1 × 10 6 A cell suspension of 1000 cells / mL was prepared and frozen in a -80°C deep freezer for 12 hours. The frozen cell solution was then placed in a 4°C refrigerator and thawed until visual confirmation of thawing was confirmed. This process was then repeated once more. The solution was stirred at 2,500 rpm in a vortex mixer for 5 minutes, and the stirred solution was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain the exosome-containing solution (for dogs).

[0070] The exosome-containing liquid (for dogs) described above was injected intravenously four times, approximately every two months, into a dog (miniature dachshund, 18 years old, female) suffering from chronic nephritis. As a result, as shown in Table 3, it was confirmed that administration of the exosome-containing liquid of the present invention reduced urinary creatinine and BUN (blood urea nitrogen) levels. Creatinine and BUN concentrations were measured using a veterinary clinical chemistry analyzer, "Fuji DryChem NX700V." [Table 3]

[0071] (3) Evaluation of exosome-containing fluid in dogs (effect on hernia) A herniated disc is a condition in which the cartilage (disc) that acts as a cushion between the vertebrae in the lumbar region of the spine degenerates, causing a protrusion of tissue, resulting in lower back pain and numbness and pain in the buttocks and legs. In dogs, herniated discs can cause symptoms such as unsteadiness when walking, tipping over of the toes, and, if the paralysis becomes severe, the dog may be unable to stand. Here, the exosome-containing liquid (for dogs) was injected intravenously into a Pomeranian (which had developed lumbar disc herniation approximately one year prior) and a miniature dachshund (which had developed disc herniation approximately three years prior) four times at weekly intervals.

[0072] The dogs' condition was observed before and after administration of the exosome-containing liquid of the present invention, and it was confirmed that walking improved, pain disappeared, and activity (vigor) increased, as shown in Table 4. The evaluations shown in Table 4 are the condition and activity state of the animals as judged by the observer (observer and / or person administering treatment) based on direct observation. The HHHHHMM Scale, a scale for measuring the quality of life of animals, was also used for evaluation.

[0073] [Table 4]

[0074] (4) Evaluation of exosome-containing fluid in dogs (activity-enhancing effect) The exosome-containing liquid (for dogs) described above was injected intravenously four times, every other week, into beagle dogs (female, 12-13 years old) with the following symptoms.

[0075] When the dogs were observed before and after administration of the exosome-containing liquid of the present invention, it was confirmed that their walking ability and activity (vigor) improved, as shown in Table 5. As with Table 4, these were the animals' appearances and activity levels judged by the observer (the observer and / or the person administering the treatment) through direct observation. It is presumed that this improvement is due to the exosomes' ability to remove reactive oxygen species and their ability to suppress inflammation and pain at the cellular level.

[0076] [Table 5] (5) Evaluation of exosome-containing fluid in horses The effect of the exosome-containing solution was also evaluated in horses. The exosome-containing solution used for the evaluation was 5 × 10 6The exosome-containing solution was prepared in the same manner as in the dog test described above, except that a cell suspension of 100 cells / mL was used. 1 mL of the exosome-containing solution was intravenously injected into racehorses, and the horses' condition was evaluated one week after administration. Evaluations were performed by veterinarians according to the following criteria: [Table 6]

[0077] The evaluation results are shown in Table 7. The results also revealed that administration of the exosome-containing liquid of the present invention improved the body condition of horses and had the effect of stabilizing their mental state. Racehorses are subject to a lot of stress from transportation, training, and races, and the effects of stress are easily apparent in areas such as coat gloss, muscle firmness, and temperament. However, administration of the exosome-containing liquid of the present invention dramatically improved body condition and temperament. [Table 7] [Industrial Applicability]

[0078] According to the production method of the present invention, it is possible to easily produce a large amount of highly concentrated exosome-containing liquid. Furthermore, pharmaceuticals and supplements containing the exosome-containing liquid of the present invention as an active ingredient are effective as agents for promoting recovery from fatigue.

Claims

1. A method for producing a mesenchymal stem cell-derived exosome-containing liquid, comprising the steps of: (1) disrupting mesenchymal stem cells; (2) stirring the disruption solution obtained in step (1) using a stirrer or a vortex mixer; (3) A step of filtering the disruption liquid after the stirring treatment.

2. The method for producing an exosome-containing liquid according to claim 1, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.

3. 3. The method for producing an exosome-containing fluid according to claim 1 or 2, wherein step (1) is carried out by freezing and thawing or ultrasonication.

4. In the step (2), 3. The method for producing an exosome-containing liquid according to claim 1 or 2, wherein the disruption liquid is stirred using a vortex mixer at 100 to 3,500 rpm for 1 to 30 minutes.

5. A pharmaceutical comprising, as an active ingredient, an exosome-containing liquid produced using the method according to claim 1 or 2.

6. A fatigue recovery promoter, wherein the pharmaceutical product according to claim 5 is administered intravenously.

7. A supplement for non-human mammals, comprising, as an active ingredient, an exosome-containing liquid produced using the method according to claim 1 or 2.

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