WO2025069570A1 - Method for producing mesenchymal stem cell-derived exosome-containing liquid, and pharmaceutical product containing exosome-containing liquid - Google Patents

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 pharmaceutical product containing exosome-containing liquid

　Related to a method for producing a liquid containing exosomes derived from mesenchymal stem cells and a pharmaceutical product containing the liquid containing exosomes.

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

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

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 regenerative medicine therapeutic effects of stem cells, and research into the use of exosomes in wound and fracture healing and in the field of regenerative medicine is being actively conducted.

In order to use exosomes in clinical practice or research, it is necessary to produce large quantities of high-quality exosomes. Methods for producing large quantities of 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 using a cell population containing mesenchymal stem cell precursors, in which 80% or more of the cells are specific marker proteins such as CD73 ⁺ (see, for example, Patent Document 2).

Patent Publication No. 2022-533277 Patent Publication No. 2022-542158

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 Emmanouilidou E. et al., “Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impact neurosurvival”, J Neurosci. 2010 May 19;30 (20): 6838-51 Ian Fyfe, “Exosomes can spread toxic AD pathology” Nat Rev Neurol. 2018 Aug;14(8) : 451 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)

The quality of exosomes is affected by the type of cells from which they are derived and the conditions under which they are produced. For this reason, it is preferable not to add additives or reagents, but the technology described in Patent Document 1 produces exosomes by adding compounds that do not exist in the body, 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 an issue in that it is a time-consuming task. Furthermore, the technology described in Patent Document 2 requires precursor cells of mesenchymal stem cells, and the method for producing the precursor cells is time-consuming and labor-intensive.

The present invention therefore aims to provide a method for easily and mass-produced high-concentration exosome-containing liquid without adding artificial compounds such as exosome production promoters.

In order to solve the above problems, the inventors focused on mesenchymal stem cells and discovered that it is possible to easily produce a large amount of highly concentrated exosome-containing liquid by disrupting the cells, which led to the completion of the present invention.

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) 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 described in [1], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.
[3] The method for producing an exosome-containing liquid described in [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 liquid according to [1] or [2], wherein the lysate 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 by the method according to [1] or [2].

　According to the production method of the present invention, it is possible to easily produce a large amount of exosome-containing liquid with a high concentration. Since expensive machines such as ultracentrifuges are not required, production costs can also be reduced. In addition, since cell fragments, rather than live cells, are used for production, the resulting exosome-containing liquid is cell-free. Therefore, the exosome-containing liquid of the present invention has a low risk of cell-derived infection or carcinogenesis, and there is no risk of causing blockage of microvessels when administered as a pharmaceutical. In addition, unlike live cells, there is no need to perform cell culture at the appropriate time for use, and the liquid is easy to prepare and handle, and can be stored for a long time by freezing. Furthermore, according to the present invention, there is no need to add additives such as exosome production promoters. Since there is no need to worry about the effects of additives on exosomes, there are great advantages in research and clinical applications.

Measurement results of exosome amount in exosome-containing liquid

<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.

Exosomes are endoplasmic reticulum secreted from cells to the outside of the cell. Exosome formation begins with the formation of early endosomes by endocytosis, engulfing receptors present on the cell membrane. The early endosome transitions to late endosomes, which then invaginate inwards to form intraluminal membrane vesicles (ILVs). Endosomes containing many ILVs (multivesicular bodies: MVBs) finally fuse with the cell membrane and are released from the cell.

The inventors focused on the mesenchymal stem cells that secrete exosomes, and by disrupting the cells, they succeeded in extracting exosomes contained within the cells before they were secreted, in addition to the secreted exosomes.

<Mesenchymal stem cells>

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 cardiac muscle cells.

Tissues containing mesenchymal stem cells include, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, 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.

　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 standard methods. Here, we will specifically explain the method for preparing adipose tissue-derived mesenchymal stem cells, which are the preferred cells.

In the present invention, the origin of the mesenchymal stem cells is not particularly limited, and includes humans and 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 administration subject (recipient) (autologous). However, this does not prevent 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>

Adipose tissue-derived mesenchymal stem cells (also known as ASC, ADSC, AT-MSC, AD-MSC, etc.) are a type of somatic stem cell found in adipose tissue. Adipose tissue-derived mesenchymal stem cells also have self-renewal and multi-differentiation abilities, 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 muscles, blood vessels, hepatocytes, and pancreatic islet cells.
<Method for preparing adipose tissue-derived mesenchymal stem cells>

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

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

ADSCs are prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix. The method for preparing ADSCs is not particularly limited. For example, ADSCs can be prepared according to known methods (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., etc. are useful references). In addition, devices for preparing ADSCs from adipose tissue (e.g., Celution (registered trademark) device (Cytori Therapeutics, San Diego, USA)) are commercially available, and ADSCs may 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 shown below.
(1) Preparation of cell populations from adipose tissue

Adipose tissue is collected from humans and non-human mammals by means of excision, suction, etc. Non-human mammals can be any of the animals mentioned above, and can include pets, livestock, and laboratory animals. There are no particular limitations on the age or sex of the organism. In humans, ADSCs can also be prepared from tissue fragments sucked out during liposuction surgery for cosmetic surgery, or from excised adipose tissue contained in tissue excised from a living body during surgery. Because ADSCs exist around large blood vessels, they can be obtained in greater quantities from excised adipose tissue than from liposuction fluid. On the other hand, preparing stem cells from liposuction fluid leaves smaller surgical scars and places a smaller burden on the donor.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat is very easy to collect under local anesthesia, which places less strain on the donor during collection and makes it a preferred cell source. Usually, one type of adipose tissue is used, but it is also possible to use two or more types of adipose tissue in combination. In addition, adipose tissue collected in multiple sessions (which do not have to be the same type of adipose tissue) can be mixed and used in subsequent operations.

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 no more than approximately 3 to 20 g at one time. The collected adipose tissue is subjected to the following enzymatic treatment after removing any blood components attached to it and cutting it into small pieces, if necessary. Blood components can be removed by washing the adipose tissue in an appropriate buffer solution or culture medium.

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 a method and under conditions known to those skilled in the art (see, for example, R. I. 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 precursor cells. The types and ratios of cells constituting the cell population depend on the origin and type of the adipose tissue used.
(2) Obtaining sedimented cell populations (SVF fractions: stromal vascular fractions)

The cell population is then subjected to centrifugation. The sediment from the centrifugation is collected as a sedimented cell population (also referred to as the "SVF fraction" in this specification). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1,500 rpm. It is preferable to filter the cell population after the enzyme treatment prior to centrifugation to remove any undigested tissues contained therein. The "SVF fraction" obtained here contains ADSC. The type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions of the enzyme treatment, etc. In addition, the characteristics of the SVF fraction are shown in the pamphlet of International Publication No. 2006/006692A1.
(3) Selective culture of adherent cells (ADSC) and cell recovery

In addition to ADSCs, the SVF fraction contains other cell components (endothelial cells, interstitial cells, blood cells, their precursor cells, etc.). Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The cells obtained as a result are 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 passages are not preferable from the viewpoint of maintaining pluripotency and proliferation ability (it is preferable to limit it to about 6 passages). Note that a medium for normal 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. can 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, 5-30% (v/v).

The above procedure allows the adhesive cells to selectively survive and proliferate. The proliferated cells are then harvested. The harvesting procedure can be performed according to conventional methods. For example, the cells can be easily harvested by detaching them with a cell scraper or pipette after enzyme treatment (trypsin or dispase treatment). In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish, it is also possible to harvest the cells in a sheet form without enzyme treatment. By using the cells (ADSC) harvested in this way, a cell population containing ADSC at high purity can be prepared.
(4) Low-serum culture (selective culture in low-serum medium) and cell recovery

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

The serum is not limited to fetal bovine serum, and 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, preferably human serum, more preferably serum from the subject to be treated (i.e. autologous serum) is used. As for the medium, a medium for normal animal cell culture can be used, provided that it contains a low amount of serum when used. 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.

By culturing using the above method, ADSCs can be selectively proliferated. Furthermore, since ADSCs proliferated 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 shows the characteristics of cells that selectively proliferate when the SVF fraction is cultured in low serum. Next, the cells selectively proliferated by the above low serum culture are collected. The collection operation can be performed in the same manner as in (3) above. By using the collected ADSCs, a cell population containing ADSCs at a high purity can be obtained.

In the above method, the cells proliferated by low serum culture of the SVF fraction are used, but the cells proliferated by directly low serum culture of a cell population obtained from adipose tissue (without centrifugation to obtain the SVF fraction) may be used as ADSCs. That is, in one aspect of the present invention, the cells proliferated when a cell population obtained from adipose tissue is cultured in low serum are used as ADSCs. In addition, the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as it is, instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)). Note that "using as it is" here means using in the present invention without selective culture.
<Method for preparing exosome-containing liquid>
(Step (1): Disintegrating ADSCs)

　ADSCs can be disrupted by a general cell disruption method. For example, methods using freeze-thaw (a process of freezing and then thawing), ultrasound, French press, mortar, homogenizer, glass beads, etc. can be used. In addition, the cells to be subjected to the disruption process are not limited to live cells, but may also be dead or damaged cells. Among the above disruption processes, freeze-thaw and ultrasound are preferred. In particular, freeze-thaw is particularly preferred because it is simple and can avoid contamination due to contact between the machine and the cells, and is hygienic. When disrupting by freeze-thaw, the preferred conditions described below can be used. When disrupting by ultrasound, it is preferable to use unfrozen cells for the disruption process, but since the heat emitted from the equipment often causes protein denaturation and aggregation, it is preferable to repeatedly perform short-term processing while cooling the cell suspension in ice. Specifically, it is preferable to repeat multiple times the process of disrupting for 5 to 15 seconds at an output of 200 W to 300 W and pausing for 10 to 30 seconds.

In addition, during the freezing process, cells expand and ice crystals form, which destroy the cells and dissolve when thawed, so it is preferable to repeat the freezing and thawing process to dissolve them sufficiently. Specifically, it is preferable to repeat the freezing and thawing process 1 to 5 times. Examples of freezing methods include subjecting the cells to a freezer, deep freezer, liquid nitrogen, etc. The freezing temperature in the freezing and thawing 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, -20°C or lower is preferable, -30°C or lower is even more preferable, and -70°C or lower is even more preferable.

There is no particular limit to the cooling time as long as the entire cell population can be frozen, but when using a freezer or freezer, it is preferable to leave it in the freezer for at least 1 hour, and preferably at least 10 hours, to allow it to cool sufficiently. There is no particular limit to the upper limit of the cooling time, but it is preferable to leave it in a refrigerator or freezer for no more than 72 hours, more preferably no more than 48 hours, and even more preferably no more than 12 hours. It is efficient to place it in a refrigerator or freezer before returning home, cool it overnight, and then work on it the next day. When using liquid nitrogen, the container containing the cells can be cooled directly or by storing the cells in a cryopreservation container and immersing the container in liquid nitrogen.

Frozen cells can be thawed by any means. For example, frozen cells can be placed in a water bath, incubator, refrigerator, thermostatic bath, or the like at a temperature higher than the freezing temperature. Specifically, thawing can be performed by leaving the cells in a refrigerator at 5°C or lower overnight, by thawing in a hot water bath (e.g., 35-40°C), or by thawing at room temperature. The concentration of the cell suspension used for disruption is preferably 1×10 ⁴ to 1×10 ⁸ cells/mL for ADSCs. More preferably, the concentration is 10×10 ⁴ to 500×10 ⁴ cells/mL for ADSCs. This is an easy-to-work-with concentration, and a sufficient amount of exosome-containing liquid can be obtained in one operation.
(Step (2): Stirring the disruption liquid)

The homogenate is placed in a container such as a test tube and stirred using a stirrer or vortex mixer. If a stirrer is used, subsequent steps such as cleaning the stirrer are required, and there is also concern that the homogenate may adhere to the stirrer and result in loss of exosomes. Therefore, stirring using a vortex mixer is preferred to increase production efficiency. The rotation speed of the vortex mixer to obtain a highly concentrated exosome-containing liquid is preferably 100 rpm to 3,500 rpm, more preferably 1,500 rpm to 2,700 rpm. The stirring time is preferably 1 to 30 minutes, more preferably 3 to 25 minutes.

By using a vortex mixer to rapidly rotate the bottom of the container and stir it, it is possible to efficiently extract exosomes from the disrupted cells, which is believed to lead to the production of a highly concentrated exosome-containing liquid. There are no particular limitations on the temperature during the stirring process.

The homogenate solution after stirring may be centrifuged, and the resulting supernatant may be used in the next step (filtering). When the homogenate solution after stirring is centrifuged, nuclei and other substances are removed from the supernatant, preventing clogging of the filter, and thus enabling efficient filter treatment. When centrifuging, it is preferable to perform centrifugation 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)

The disrupted liquid after stirring or the supernatant obtained by centrifugation is filtered. Unnecessary components can be removed by filtering. Furthermore, if a filter with an appropriate pore size is used, removal of unnecessary components and sterile filtration can be performed simultaneously. There are no particular restrictions on the material of the filter used for filtering, 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. 0.15 to 0.3 μm is even more preferred. If sterile filtration is also performed at the same time, a pore size of 0.2 μm is preferred.

If the filtrate is not to be used immediately for treatment, it can be frozen and stored until the time of use. It is preferable to store it at -100 to -60°C. In general, repeated freezing and thawing of cells tends to reduce the activity of the cells and increase the number of dead cells, but since the exosome-containing liquid of the present invention does not contain stem cells, its quality will not change no matter how many times it is frozen and thawed.

The exosome-containing liquid of the present invention can be used as a raw material without isolating the exosomes, but the exosomes can also 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 of these. Note that exosomes may be separated from the exosome-containing liquid using an ultracentrifuge, although this is an expensive machine.

Confirmation that the exosomes obtained in the present invention are the intended ones can be performed 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>

The exosome-containing liquid of the present invention preferably contains 10 to 10,000 pg/mL of exosomes, 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 the amount of CD63 protein using a CD9 x CD63 ELISA kit for quantifying human exosomes manufactured by Cosmo Bio Co., Ltd.

　According to the production method of the present invention, in addition to exosomes secreted outside the cells, exosomes contained within the cells can be efficiently extracted. Therefore, a highly concentrated exosome-containing liquid can be produced. Furthermore, since no expensive machinery such as an ultracentrifuge is required and the operation is very simple, production costs can be reduced.

In addition, since the exosome-containing liquid of the present invention does not contain live cells, there is little risk of carcinogenesis or other issues caused by live cells, and there is no problem of transplant rejection, and there is no risk of clogging microvessels when administered as a pharmaceutical. It is easy to prepare and handle, and can be stored for a long time by freezing. Furthermore, the exosome-containing liquid of the present invention not only contains a large amount of exosomes, but also contains a large amount of various growth factors contained within cells.
<Uses of the exosome-containing liquid of the present invention>

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, which will be described later. 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.

For example, when used as a pharmaceutical, the pharmaceutical includes a pharmaceutical comprising, as an active ingredient, the exosome-containing liquid or separated exosome 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 is effective for tissue regeneration because it is presumed to contain not only a large amount of exosomes but also a large amount of various growth factors.

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 function disorders, 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.
In addition, the exosome-containing liquid of the present invention is presumed to have the effects of 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 conditions, improving the developmental ability 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 reduced function of eggs or sperm, oligospermia, endometriosis, etc., to improve the success rate of in vitro fertilization, and to breed livestock and maintain breeding and species (for example, to maintain endangered species, maintain or crossbreed pet lineages), etc.
In addition, due to their lipid bilayer structure, exosomes are said to be able to penetrate into the dermis layer of the skin and exert skin regeneration and anti-aging effects by increasing and activating fibroblasts, which are cells that compose the skin, as well as promoting collagen synthesis. Therefore, they can also be used for anti-aging and cosmetic purposes.

　A pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient may contain non-toxic, inactive, medicamentously 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, flavorings, colorants, fragrances, preservatives, antibacterial agents, media, saline, antibacterial agents, and drugs with other medicinal properties may be contained as additives.

In particular, it is preferable that the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient contains an antibacterial agent. An antibacterial agent refers to a compound with antibacterial activity, and specific examples include Antibiotic-Antimycotic Solution, isopropyl methylphenol, ginger oil, thymol, chlorhexidine hydrochloride, cetylpyridinium chloride, etc. These antibacterial agents are preferably contained in an effective amount to exert antibacterial activity. By containing an antibacterial agent in the pharmaceutical of the present invention, the proliferation of infectious bacteria is suppressed and safety is guaranteed for a certain storage period.

The dosage form of the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient is not particularly limited, and may be liquid or solid, or may be processed into a powder form by freezing and/or drying, and may be formulated in various ways depending on the purpose of use. The dosage form may be a powder, granule, tablet, capsule, injection, drip, inhalant, ointment, eye drop, nasal drop, or transdermal absorption agent.

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. Examples include subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, local direct injection, direct application, etc.

The dosage of the pharmaceutical containing the exosome-containing liquid of the present invention as an active ingredient may be any amount that provides a therapeutic effect, and an effective amount is administered. The level depends on the individual type, severity, age, sex, type of disease, etc. The frequency of administration is such that a therapeutic effect can be obtained for the disease when the pharmaceutical of the present invention is administered. The period of administration is such that a therapeutic effect can be obtained for the disease when the pharmaceutical of the present invention is administered.

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

The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples in any way.
(1) Preparation of exosome-containing solution <Preparation of ADSC lysate filtrate (freeze-thaw)>

A filtrate was prepared using human adipose stem cells by the method described herein. Specifically, the human adipose stem cells were cultured in Messenger Pro RS medium to proliferate ADSCs. The cells were subcultured for 6 passages until 5×10 ⁶ cells were obtained, with the medium replaced every 2 days. The concentration was adjusted using PBS to prepare a cell suspension of 1×10 ⁶ cells/mL. These were frozen in a freezer at -20°C for 1 day and in a deep freezer at -80°C for 12 hours. Liquid nitrogen (-196°C) can freeze instantly, but here the container containing the cell suspension was immersed in liquid nitrogen for 30 minutes before the start of the measurement.

The frozen cell solution was then placed in a refrigerator at 4°C and thawed until thawing was confirmed by visual inspection. After disrupting the cells in this way, the solution was stirred using a vortex mixer for the stirring time listed in Table 1. The rotation speed was 2,500 rpm. The solution after stirring 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

The amount of exosomes in the exosome-containing solution, culture supernatant, and serum-free medium prepared above was measured as the amount of CD63 protein using a CD9 x CD63 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.

As is clear from Table 1 and Figure 1, the exosome-containing liquid obtained by vortexing the frozen and 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.

As described above, according to the production method of the present invention, a high-concentration exosome-containing liquid can be produced in a large amount and easily. It is expected that exosomes can be used for various purposes.

(2) Effect of exosome-containing fluid on chronic nephritis Exosome-containing fluid was prepared from canine mesenchymal stem cells (beagle, 3-year-old male) in the same manner as for human adipose stem cells. Note that the canine cells used met the following requirements:
- Receive combined vaccinations, heartworm prevention and rabies vaccinations every year,
- Have 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 severe metabolic or endocrine disorders
- Not suffering from any genetic disease,
- Not suffering from malignant tumors,
Canine ADSCs that met the above requirements were collected, expanded, and subcultured for six passages to prepare a cell suspension of 1 x ¹⁰⁶ cells/mL. The cells were 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 thawing was confirmed by visual inspection. This process was then repeated once more. The solution was stirred at 2,500 rpm for 5 minutes in a vortex mixer, and the solution after the stirring process was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain the exosome-containing solution (for dogs).

The above-mentioned exosome-containing liquid (for dogs 1) was injected intravenously into a dog (miniature dachshund, 18 years old, female) suffering from chronic nephritis, a total of four times at intervals of about two months. As a result, as shown in Table 2, it was confirmed that the urinary creatinine and BUN (urea nitrogen) values were reduced by administering the exosome-containing liquid of the present invention. The concentrations of creatinine and BUN were measured using a veterinary clinical chemistry analyzer "Fuji DryChem NX700V".

(3) Effect of exosome-containing fluid on herniation Intervertebral disc herniation is a disease in which the cartilage (intervertebral disc), which acts as a cushion between the vertebrae in the lumbar region of the spine, degenerates and a part of the tissue protrudes, causing lower back pain and numbness and pain in the buttocks and legs. When intervertebral disc herniation occurs in dogs, symptoms include unsteadiness when walking, tipping over of the toes, and in severe paralysis, being unable to stand up.
Here, the exosome-containing liquid (for dogs) was injected intravenously into a Pomeranian (which had developed lumbar disc herniation approximately one year ago) and a miniature dachshund (which had developed disc herniation approximately three years ago) four times at weekly intervals.

When the dogs were observed before and after administration of the exosome-containing liquid of the present invention, 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 animals' appearance and activity state judged by the observer (the observer and/or the person administering the treatment) through direct observation. The HHHHHMM Scale, which measures the quality of life of animals, was also used in the evaluation.

(3) Activity-Enhancing Effect of Exosome-Containing Fluid The above-mentioned exosome-containing fluid (for dogs) was injected intravenously four times at weekly intervals to beagle dogs (female, 12 to 13 years old) with the following symptoms.

When the dogs were observed before and after administration of the exosome-containing liquid of the present invention, it was confirmed that their walking improved and their activity (vigor) increased, as shown in Table 4. Note that, like Table 3, this is the state and activity of the animals judged by the observer (the observer and/or the person administering the treatment) upon 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.

The production method of the present invention makes it possible to easily produce a large amount of highly concentrated exosome-containing liquid.

Claims (5)

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 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. The method for producing an exosome-containing liquid according to claim 1, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. The method for producing an exosome-containing liquid according to claim 1 or 2, wherein step (1) is carried out by freezing and thawing or ultrasonication. 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 at 100 to 3,500 rpm using a vortex mixer for 1 to 30 minutes. 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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