Method for producing mesenchymal stem cell-derived exosome-containing liquid, and pharmaceutical product containing exosome-containing liquid

Abstract

[Problem] To provide a method for producing a high-concentration exosome-containing liquid in a large amount and in a simple manner. [Solution] The present inventors have found that a high-concentration exosome-containing liquid can be produced in a large amount and in a simple manner by crushing mesenchymal stem cells. Specifically, the present invention comprises: (1) a step for crushing mesenchymal stem cells; (2) a step for stirring the crushed liquid obtained in the step (1) using a stirrer or a vortex mixer; and (3) a step for filtering the stirred crushed liquid with a filter. An exosome-containing liquid produced in the present invention is cell-free and is thus easily prepared and handled and can also be stored for a long period through freezing. Moreover, since an additive is not added, there is no need to worry about the influence of the additive on exosomes, and the research and clinical advantages are extremely significant.

Description

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

[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.

[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 contain 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 culture medium containing progenitor cells of mesenchymal stem cells, 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).

[0006] Special Publication No. 2022-533277 Publication Special Publication No. 2022-542158

[0007] 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) 2739-2749Emmanouilidou E. et al., “Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and neuronal impacts 2010 May 19;30 (20) : 6838-51Ian Fyfe, “Exosomes can spread toxic AD pathology” Nat Rev Neurol. 2018 Aug;14(8) : 451Yamayoshi A., et al., “Development of Novel Drug Delivery System Targeting Exosomal microRNA” The Pharmaceutical Society of Japan, Vol. 140, No.5, 625-631 (2020)

[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.

[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 configurations. [1] A method for producing an exosome-containing liquid derived from mesenchymal stem cells, comprising the following steps: (1) disrupting mesenchymal stem cells, (2) stirring the disrupted liquid obtained in step (1) using a stirrer or a vortex mixer, and (3) filtering the disrupted 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 liquid according to [1] or [2], wherein step (1) is carried out by freezing and thawing or ultrasonication. [4] The method for producing an exosome-containing liquid according to [1] or [2], wherein in step (2), the disrupted liquid is stirred using a vortex mixer at 100 to 3,500 rpm for 1 to 30 minutes. [5] A pharmaceutical product comprising, as an active ingredient, an exosome-containing liquid produced using the method according to [1] or [2].

[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 an ultracentrifuge is not required, production costs can be reduced. Furthermore, because cell fragments, rather than live cells, are used for production, the resulting exosome-containing solution is cell-free. Therefore, the exosome-containing solution of the present invention poses little risk of cell-derived infection or carcinogenesis, and there is no risk of microvascular blockage when administered as a pharmaceutical. Furthermore, unlike live cells, there is no need to carefully time cell culture 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, it offers significant advantages in research and clinical applications.

[0013] Measurement results of exosome amount in exosome-containing liquid

[0014] <Exosome> 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 encapsulates 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 origin of the mesenchymal stem cells in the present invention is not particularly limited, and includes humans as well as non-human mammals (including pet animals, livestock, and laboratory animals, specifically, for example, 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 (ASC), Adipose-derived regeneration cells (ADSC), Adipose-derived mesenchymal stem cells (AT-MSC), AD-MSC, etc.; hereinafter, simply referred to as "ADSC") are a type of somatic stem cell found in adipose tissue. Adipose tissue-derived mesenchymal stem cells also have the ability to self-renew and multilineage differentiation, 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 refer to cells obtained by culturing (including subculture) somatic stem cells, as long as they maintain pluripotency. ADSCs are usually prepared in an "isolated state" from 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). In addition, devices for preparing ADSCs from adipose tissue (e.g., the Celution (registered trademark) 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). Specific examples of methods for preparing ADSCs are shown below. (1) Preparation of a cell population 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, minimizing the burden 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. Furthermore, adipose tissues collected in multiple batches (which do not have to be the same type of adipose tissue) can be mixed and used for subsequent procedures.

[0028] The amount of adipose tissue to be collected can be determined taking into account 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 attached thereto 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] Enzyme treatment is carried out by digesting the adipose tissue with enzymes such as collagenase, trypsin, and dispase. Such enzyme treatment may be carried out by methods 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 includes 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, it is preferable to filter the cell population after enzyme treatment to remove any undigested tissues contained therein. The "SVF fraction" obtained here contains ADSCs. The types and ratios 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 also described in International Publication No. 2006 / 006692A1. (3) Selective Culture of Adherent Cells (ADSCs) and Cell Recovery

[0031] The SVF fraction contains ADSCs as well as other cellular components (endothelial cells, stromal cells, blood cells, and 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 resulting cells are then used as ADSCs in the present invention. First, the SVF fraction is suspended in an appropriate medium, seeded on a culture dish, and cultured overnight. Non-adherent cells are removed by changing the medium. Culture is then continued while changing the medium as appropriate (e.g., every 2–4 days). Subculture is performed as needed. While the number of passages is not particularly limited, excessive passages are not recommended from the perspective of maintaining pluripotency and proliferation ability (preferably limited to approximately 6 passages). Conventional animal cell culture media 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 (knockout serum replacement (KSR), etc.) 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 allows the adherent cells to selectively survive and proliferate. The proliferated cells are then recovered. The recovery procedure can be performed according to conventional methods. For example, cells can be easily recovered by detaching them with a cell scraper or pipette after enzyme treatment (trypsin or dispase treatment). Furthermore, when sheet culture is performed using commercially available temperature-sensitive culture dishes, it is also possible to recover the cells in a sheet form without enzyme treatment. Using the cells (ADSCs) recovered in this way, a cell population containing highly pure 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, autologous serum can be used for culture. Here, "low-serum conditions" refers to conditions in which the medium contains 5% (v / v) or less serum. Preferably, cells are cultured in a culture medium containing 2% (v / v) or less serum. More preferably, cells are cultured in a culture medium containing 2% (v / v) or less serum and 1-100 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. Examples of media that can be used include 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.), and MCDB201 medium (Functional Peptide Institute).

[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 / 006692 A1 describes the characteristics of cells that selectively proliferate when cultured in low serum from the SVF fraction. 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. 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 utilized. However, cells proliferated by directly low-serum culture of a cell population obtained from adipose tissue (without the centrifugation step to obtain the SVF fraction) may also be used as ADSCs. That is, in one embodiment 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): Step of 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, French press, mortar, 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 avoids contamination due to contact between the machine and the cells, making it hygienic. 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, it is preferable to repeat the freeze-thaw process 1 to 5 times. Examples of freezing methods include using a freezer, deep freezer, or liquid nitrogen. The freezing temperature in the freeze-thaw process is not particularly limited as long as it is a temperature that can freeze the entire cell population. A temperature between -20°C and -196°C is preferable, but more specifically, a temperature of -20°C or lower is preferable, -30°C or lower is even more preferable, and -70°C or lower is even more preferable.

[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 hot 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 ~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 lysate)

[0041] The homogenized solution 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 homogenized solution 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.

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

[0044] The disrupted solution after stirring or the supernatant obtained by centrifugation is filtered. Unnecessary components can be removed by filtration. Furthermore, if a filter with an appropriate pore size is used, removal of unnecessary components and sterile filtration can be performed simultaneously. The material of the filter used for filtration is not particularly limited, but cellulose acetate and 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.

[0045] If the filtrate is not to be used immediately for treatment, it can be frozen and stored until use. Storage at -100 to -60°C is preferable. 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] The exosomes obtained by the present invention can be confirmed to be the intended ones by morphological observation using a transmission electron microscope, gene analysis using PCR, immunological techniques (ELISA, FACS, etc.), Western blotting, etc. <Exosome-containing liquid of the present invention>

[0048] The exosome-containing solution of the present invention preferably contains exosomes at a concentration of 10 to 10,000 pg / mL, more preferably 50 to 7,500 pg / mL, more preferably 75 to 5,000 pg / mL, even more preferably 100 to 1,000 pg / mL, and even more preferably 100 to 500 pg / mL, when measured as CD63 protein using a CD9×CD63 ELISA kit for quantifying human exosomes manufactured by Cosmo Bio Co., Ltd.

[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, since the exosome-containing liquid of the present invention does not contain live cells, there is little risk of carcinogenesis or other problems associated with live cells, and there is no problem of transplant rejection. Furthermore, there is no risk of microvascular blockage when administered as a pharmaceutical. Preparation and handling are easy, and long-term storage by freezing is also possible. 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 and the like 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, 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 or the like.

[0052] For example, when used as a pharmaceutical, the exosome-containing liquid or isolated exosomes obtained by the production method of the present invention may be used as an active ingredient. 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 is effective for tissue regeneration because it not only contains a large amount of exosomes but also is thought to contain large amounts of various growth factors.

[0053] Specifically, the exosome-containing solution of the present invention 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 or motor dysfunction, such as rheumatism, osteoarthritis of the knee, hernias and disorders that cause difficulty in maintaining balance, and diseases that cause muscle weakness such as amyotrophic lateral sclerosis. Furthermore, the exosome-containing solution of the present invention is expected to have effects such as 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, so it can be used to treat or improve infertility caused by decreased egg or sperm function, oligospermia, endometriosis, etc., to improve the success rate of in vitro fertilization, livestock breeding, breeding and species maintenance (e.g., preserving endangered species, maintaining or crossbreeding pet breeds), etc. Furthermore, because of their lipid bilayer structure, exosomes are said to penetrate into the dermis layer of the skin, resulting in skin regeneration and anti-aging effects due to the proliferation and activation of fibroblasts, which are skin constituent cells, and to promote collagen synthesis, so they can also be used for anti-aging and cosmetic purposes.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The method of administration of the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient is not particularly limited, and may be local administration or systemic administration, such as subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, local direct injection, or direct application.

[0058] 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.

[0059] 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.

[0060] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. (1) Preparation of exosome-containing solution <Preparation of ADSC lysate (freeze-thawed)>

[0061] A filtrate was prepared using human adipose stem cells according to the method described herein. Specifically, the human adipose stem cells were cultured in Messenger Pro RS medium to proliferate ADSCs. The cells were cultured at a concentration of 5 × 10 with medium changes every two days. 6 The cells were subcultured for 6 passages until the cells were obtained. The concentration was adjusted using PBS and the cells were diluted to 1 × 10 6 A cell suspension of 1000 cells / mL was prepared. It was frozen in a -20°C freezer for one day and in a -80°C deep freezer for 12 hours. Liquid nitrogen (-196°C) can freeze cells instantly, but here the container containing the cell suspension was immersed in liquid nitrogen for 30 minutes before the start of the measurement.

[0062] The frozen cell solution was then placed in a refrigerator at 4°C and thawed until thawing was confirmed visually. After disrupting the cells in this manner, the solution was stirred using a vortex mixer for the stirring time listed in Table 1. The rotation speed was 2,500 rpm. The stirred solution was then filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain an exosome-containing solution. Serum-free medium (KBM ADSC-5, Kohjin Bio Co., Ltd.) was used as a control. <Quantitative analysis of exosomes by ELISA>

[0063] The amount of exosomes in the exosome-containing solution, culture supernatant, and serum-free medium prepared above was measured as CD63 protein using a CD9xCD63 ELISA kit for quantifying human-derived exosomes manufactured by Cosmo Bio Co., Ltd. The measurement method followed the kit manufacturer's protocol. The results are shown in Table 1 and Figure 1. Figure 1 is a graph of the results in Table 1.

[0064] As is clear from Table 1 and Figure 1, the exosome-containing solution obtained by vortexing the frozen-thawed sample and filtering it contained a high concentration of exosomes. The effect was more pronounced the lower the freezing temperature. In particular, compared to the control (culture supernatant), Example 3-3 contained approximately 30 times as much exosomes.

[0065] As described above, the production method of the present invention makes it possible to easily produce a large amount of exosome-containing liquid at a high concentration, and is expected to enable the use of exosomes in a variety of applications.

[0066] (2) Effect of exosome-containing solution on chronic nephritis Exosome-containing solution was prepared using canine mesenchymal stem cells (beagle, 3-year-old male) in the same manner as human adipose stem cells. The cells used were from dogs that met the following requirements: ・Received combined vaccinations, heartworm prevention, and rabies vaccinations annually; ・Has not received a blood transfusion in the past; ・Has no systemic infectious skin disease; ・Has not previously contracted or been suspected of having contracted a blood-borne infectious disease (such as babesiosis or canine brucellosis); ・Has no serious metabolic or endocrine disorders; ・Has no genetic disorders; ・Has no malignant tumors. ADSCs from dogs that met the above requirements were collected, expanded, and cultured for 6 passages, resulting in a total of 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 suspension 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 suspension was then vortexed at 2,500 rpm for 5 minutes, and the resulting solution was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain the exosome-containing solution (for dogs).

[0067] The exosome-containing solution (for dogs, 1) 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 2, it was confirmed that administration of the exosome-containing solution of the present invention reduced urinary creatinine and BUN (blood urea nitrogen). Creatinine and BUN concentrations were measured using a veterinary clinical chemistry analyzer, "Fuji DryChem NX700V."

[0068] (3) Effect of exosome-containing liquid on herniation. Intervertebral disc herniation is a condition in which the cartilage (intervertebral disc), which acts as a cushion between the vertebrae in the lumbar region of the spine, degenerates and causes a protrusion of tissue, resulting in lower back pain and numbness and pain in the buttocks and legs. In dogs, disc herniation can cause symptoms such as unsteadiness when walking, tipping over of the toes, and, if the paralysis worsens, inability to stand. Here, the exosome-containing liquid (for dogs) was injected intravenously into a Pomeranian (which had developed lumbar disc herniation approximately one year earlier) and a miniature dachshund (which had developed disc herniation approximately three years earlier) four times at weekly intervals.

[0069] 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 3. The evaluations shown in Table 3 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.

[0070]

[0071] (3) Activity-enhancing effect of exosome-containing liquid Beagle dogs (female, 12-13 years old) with the following symptoms were intravenously injected with the exosome-containing liquid (for dogs) described above four times at weekly intervals.

[0072] 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 4. As with Table 3, these results were based on the animals' condition and activity state as judged by the observer (the observer and / or the person administering the treatment) through direct observation. This improvement is presumed to be due to the exosomes' ability to remove reactive oxygen species and their ability to suppress inflammation and pain at the cellular level.

[0073]

[0074] According to the production method of the present invention, a highly concentrated exosome-containing liquid can be produced in a large amount and easily.

Claims

1. A method for producing a liquid containing exosomes derived from mesenchymal stem cells, comprising the steps of: (1) disrupting mesenchymal stem cells; (2) stirring the disrupted liquid obtained in step (1) using a stirrer or a vortex mixer; and (3) filtering the disrupted 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. The method for producing an exosome-containing liquid according to claim 1 or 2, wherein the step (1) is carried out by freezing and thawing or ultrasonication.

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

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