JP7478227B2 - Cell aging inhibitor, biological tissue repair promoter, gene expression regulator, and manufacturing method - Google Patents

Description

The present invention relates to a cellular aging inhibitor, a biological tissue repair promoter, and a gene expression regulator using extracellular vesicles derived from mesenchymal stem cells.

Cellular aging is a phenomenon in which cells stop dividing, the production of extracellular matrix decreases, or factors that damage surrounding tissues are released. Living tissues are maintained by the proliferation of individual cells and the production of extracellular matrix, so cellular aging has a significant impact on the deterioration of tissues. For example, when DNA is damaged due to the accumulation of DNA replication errors caused by cell division, oxidative stress, radiation, activation of cancer genes, etc., the expression of cell proliferation inhibitory genes such as p53 gene, p21 gene, and p16 gene is enhanced by the DNA damage response, which is thought to cause cellular aging. In addition, for example, the skin, which is composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen, hyaluronic acid, and elastin, is thought to have symptoms such as wrinkles, sagging, and age spots due to the decline in the function of the extracellular matrix and fibroblasts in particular due to ultraviolet rays, dryness, stress, aging, etc.

Extracellular vesicles, which are small membrane vesicles composed of a lipid bilayer membrane derived from cells, are thought to be responsible for intercellular signaling through the transport of nucleic acids such as mRNA and microRNA, or proteins, which they encapsulate (Non-Patent Document 1).

Mesenchymal stem cells (MSCs) are stem cells that have the ability to differentiate into cells belonging to mesodermally derived tissues (mesenchymal system). MSCs can be isolated from fat, bone marrow, umbilical cord matrix, etc., and are generally considered to have the following common features: they are adhesive; they are positive for CD105, CD73, and CD90; they are negative for CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA-Class II (DR); and they have the ability to differentiate into bone, fat, and cartilage.

In recent years, it has been suggested that extracellular vesicles may be involved in various diseases. It has also been reported that extracellular vesicles derived from mesenchymal stem cells obtained by ultracentrifugation promote cell proliferation (Non-Patent Document 2), dermis repair (Non-Patent Document 3), and ligament repair (Non-Patent Document 4).

International Publication WO2016/088689

Mathieu M, Martin-Jaular L, Lavieu G, Thery C (2019) Nat Cell Biol, 21(1): 9-17 Arsalán S et al. STEM CELLS AND DEVELOPMENT Volume 24, Number 14, 2015 Bocheng Zhang et al. Cell Transplantation Volume 29:1-14 Connie S. Chamberlain et al. Stem Cells. 2021;39:55-61.

However, when the present inventors examined the cellular aging inhibitory activity and biological tissue repair promoting activity of extracellular vesicles derived from mesenchymal stem cells obtained by ultracentrifugation, they found that these activities were weak and insufficient for industrial use. In view of the above situation, an object of the present invention is to provide a cellular aging inhibitor, a biological tissue repair promoter, a gene expression regulator, and a skin topical composition composed of a population of extracellular vesicles having higher cellular aging inhibitory activity and biological tissue repair promoting activity.

In order to solve the above problems, the present inventors conducted extensive research into extracellular vesicles selectively recovered by various methods for obtaining extracellular vesicles, and as a result, discovered that a population of extracellular vesicles derived from mesenchymal stem cells stimulated with inflammatory cytokines and/or a population of extracellular vesicles characterized by being PS-positive, obtained by a method utilizing a substance having affinity for phosphatidylserine (PS) (PS affinity method), have high cell aging inhibitory activity, high gene expression modulatory activity as a gene expression modulator, and high tissue repair activity as a tissue repair promoter, thereby completing the present invention.

That is, the basic aspect of the present invention is
(1) A cellular aging inhibitor or a biological tissue repair promoter, comprising extracellular vesicles derived from mesenchymal stem cells as active ingredients,
A cellular aging inhibitor or a biological tissue repair promoter, wherein the extracellular vesicles are derived from mesenchymal stem cells stimulated with inflammatory cytokines, or/and the extracellular vesicles are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(2) The cell aging inhibitor or biological tissue repair promoter according to (1) above, wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane, and placenta;
(3) The cell aging inhibitor or biological tissue repair promoter according to (1) or (2) above, wherein the mesenchymal stem cells are stimulated with at least one inflammatory cytokine selected from tumor necrosis factor α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ;
(4) The cell aging inhibitor or biological tissue repair promoter according to any one of (1) to (3) above, wherein the extracellular vesicles are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(5) The cell aging inhibitor or biological tissue repair promoter according to (4) above, wherein the substance having affinity for phosphatidylserine is a Tim protein;
(6) The cell aging inhibitor or biological tissue repair promoter according to (5) above, wherein the Tim protein is selected from the group consisting of Tim4 protein, Tim3 protein, and Tim1 protein;
(7) The agent for inhibiting cell aging or promoting the repair of biological tissue according to (1) or (2) above, wherein the extracellular vesicles are derived from mesenchymal stem cells stimulated with an inflammatory cytokine and are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(8) The cellular aging inhibitor or biological tissue repair promoter according to any one of (1) to (7) above, wherein the cellular aging inhibition or biological tissue repair promotion is due to at least one action selected from a cell proliferation promoting action, a collagen production promoting action or a collagen degradation inhibiting action, a hyaluronic acid production promoting action or a collagen degradation inhibiting action, and an elastin production promoting action or a collagen degradation inhibiting action;
It is.

The present invention also relates to a method for producing extracellular vesicles as such a cell aging inhibitor or a biological tissue repair promoter, specifically, the method comprising:
(9) A method for producing extracellular vesicles having an effect of inhibiting cellular aging or an effect of promoting the repair of biological tissue, comprising obtaining extracellular vesicles from mesenchymal stem cells stimulated with inflammatory cytokines, and/or obtaining extracellular vesicles from extracellular vesicles derived from mesenchymal stem cells by a method using a substance having affinity for phosphatidylserine;
(10) The method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to (9) above, wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane, and placenta;
(11) The method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to (9) or (10) above, wherein the mesenchymal stem cells are stimulated with at least one inflammatory cytokine selected from tumor necrosis factor α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ;
(12) A method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repairing effect according to any one of (9) to (11) above, wherein the extracellular vesicles are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(13) The method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to any one of (9) to (12) above, wherein the substance having affinity for phosphatidylserine is a Tim protein;
(14) The method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to (13) above, wherein the Tim protein is selected from the group consisting of Tim4 protein, Tim3 protein, and Tim1 protein;
(15) The method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to (9) or (10) above, wherein the extracellular vesicles are derived from mesenchymal stem cells stimulated with inflammatory cytokines and are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(16) A method for producing extracellular vesicles having a cellular aging inhibitory effect or a biological tissue repair promoting effect according to any one of (9) to (15) above, wherein the cellular aging inhibitory effect or biological tissue repair promoting effect is due to at least one effect selected from the group consisting of an effect of promoting collagen production or inhibiting decomposition, an effect of promoting hyaluronic acid production or inhibiting decomposition, and an effect of promoting elastin production or inhibiting decomposition;
It is.

The present invention also relates to a gene expression regulator, specifically,
(17) A gene expression regulator comprising extracellular vesicles derived from mesenchymal stem cells as active ingredients, the extracellular vesicles being derived from mesenchymal stem cells stimulated with inflammatory cytokines, or/and the extracellular vesicles being obtained by a method utilizing a substance having affinity for phosphatidylserine, and the gene being at least one kind of gene selected from a cell proliferation inhibitory gene, a collagen gene, a collagen decomposition enzyme gene, a hyaluronic acid synthase gene, a hyaluronic acid decomposition enzyme gene, an elastin gene, and an elastin decomposition enzyme gene;
It is.

The present invention also relates to a method for producing extracellular vesicles as gene expression regulators, specifically, the method comprising the steps of:
(18) A method for producing extracellular vesicles having a gene expression regulating effect, comprising obtaining extracellular vesicles from mesenchymal stem cells stimulated with inflammatory cytokines, and/or obtaining extracellular vesicles from extracellular vesicles derived from mesenchymal stem cells by a method using a substance having affinity for phosphatidylserine;
(19) The method for producing extracellular vesicles having a gene expression-regulating activity according to (18) above, wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane and placenta;
(20) The method for producing extracellular vesicles having a gene expression-regulating activity according to (18) or (19) above, wherein the mesenchymal stem cells are stimulated with at least one inflammatory cytokine selected from tumor necrosis factor α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ;
(21) The method for producing extracellular vesicles having a gene expression regulating activity according to any one of (18) to (20) above, wherein the extracellular vesicles are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(22) The method for producing extracellular vesicles having a gene expression regulating activity according to (21) above, wherein the substance having affinity for phosphatidylserine is a Tim protein;
(23) The method for producing extracellular vesicles having a gene expression regulating activity according to (22) above, wherein the Tim protein is selected from the group consisting of Tim4 protein, Tim3 protein and Tim1 protein;
(24) A method for producing extracellular vesicles having a gene expression regulating activity according to any one of (18) to (20) above, wherein the extracellular vesicles are derived from mesenchymal stem cells stimulated with inflammatory cytokines and are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(25) The method for producing extracellular vesicles having a gene expression regulating effect according to any one of (18) to (24) above, wherein the gene is at least one gene selected from a cell proliferation inhibitory gene, a collagen gene, a collagen decomposition enzyme gene, a hyaluronic acid synthase gene, a hyaluronic acid decomposition enzyme gene, an elastin gene, and an elastin decomposition enzyme gene;
It is.

The present invention also relates to a composition for external application to the skin for inhibiting cellular aging or promoting the repair of biological tissue, specifically comprising:
(26) A composition for external application to the skin for inhibiting cell aging or promoting the repair of biological tissue, comprising the cell aging inhibitor or the biological tissue repair promoter according to any one of (1) to (8) above;
It is.

The present invention also relates to a method for producing a composition for external application to the skin for inhibiting cellular aging or promoting biological tissue repair, which specifically comprises the steps of:
(27) A method for producing an external composition for skin for inhibiting cellular aging or promoting the repair of biological tissue, comprising obtaining extracellular vesicles from mesenchymal stem cells stimulated with inflammatory cytokines, and/or obtaining extracellular vesicles from extracellular vesicles derived from mesenchymal stem cells by a method using a substance having affinity for phosphatidylserine;
(28) The method for producing the composition for external application to skin for inhibiting cellular aging or promoting the repair of biological tissue according to (27) above, wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane and placenta;
(29) The method for producing an external composition for skin for inhibiting cellular aging or promoting biological tissue repair according to (27) or (28) above, wherein the mesenchymal stem cells are stimulated with at least one inflammatory cytokine selected from tumor necrosis factor α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ;
(30) A method for producing a composition for external use on skin for inhibiting cellular aging or promoting the repair of biological tissue according to any one of (27) to (29) above, wherein the extracellular vesicles are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(31) The method for producing an external composition for skin for inhibiting cellular aging or promoting biological tissue repair according to any one of (27) to (30) above, wherein the substance having affinity for phosphatidylserine is a Tim protein;
(32) The method for producing an external composition for skin for inhibiting cellular aging or promoting biological tissue repair according to (31) above, wherein the Tim protein is selected from the group consisting of Tim4 protein, Tim3 protein and Tim1 protein;
(33) A method for producing a composition for external use on skin for inhibiting cellular aging or promoting the repair of biological tissue according to any one of (27) to (29) above, wherein the extracellular vesicles are derived from mesenchymal stem cells stimulated with inflammatory cytokines and are obtained by a method utilizing a substance having affinity for phosphatidylserine;
(34) A method for producing a composition for external use on the skin for inhibiting cellular aging or promoting biological tissue repair according to any one of (27) to (33) above, wherein the cellular aging inhibitory effect or biological tissue repair promoting effect is due to at least one effect selected from the group consisting of an effect of promoting collagen production or inhibiting collagen decomposition, an effect of promoting hyaluronic acid production or inhibiting collagen decomposition, and an effect of promoting elastin production or inhibiting collagen decomposition;
It is.

The present invention provides a cell aging inhibitor, a biological tissue repair promoter, a gene expression regulator, and a skin topical composition, which contain as active ingredients extracellular vesicles having a cell aging inhibitory effect or a biological tissue repair promoting effect from mesenchymal stem cells. The cell aging inhibitor, biological tissue repair promoter, and skin topical composition provided by the present invention regulate the gene expression of a cell aging-related gene or a biological tissue repair-related gene, and inhibit cell aging and promote the repair of biological tissue by promoting cell proliferation, promoting collagen production or inhibiting the degradation, promoting hyaluronic acid production or inhibiting the degradation, promoting elastin production or inhibiting the degradation, etc. In addition, the gene expression regulator provided by the present invention has an expression regulator effect on at least one gene selected from a cell proliferation inhibitory gene, a collagen gene, a collagen decomposition enzyme gene, a hyaluronic acid synthase gene, a hyaluronic acid decomposition enzyme gene, an elastin gene, and an elastin decomposition enzyme gene.

FIG. 1 shows the results of NTA analysis of particle size distribution of MSC-derived extracellular vesicles obtained by ultracentrifugation. FIG. 2 shows the results of NTA analysis of the particle size distribution of MSC-derived extracellular vesicles obtained by the PS affinity method. FIG. 3 shows the primers used in quantitative PCR. FIG. 4 shows the results of evaluating the cell aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from bone marrow-derived MSCs using human fetal lung fibroblasts (TIG3 cells) and the mRNA expression level of the p21 gene (a cell proliferation-inhibiting gene) as an indicator. FIG. 5 shows the results of evaluating the cell aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from bone marrow-derived MSCs using human fetal lung-derived fibroblasts and the mRNA expression level of the p53 gene (a cell proliferation-inhibiting gene) as an indicator. Figure 6 shows the results of evaluating the cellular aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from bone marrow-derived MSCs using human fetal lung-derived fibroblasts and the mRNA expression level of the MMP-1 gene (collagen-degrading enzyme gene) as an indicator. FIG. 7 shows the results of evaluating the cell aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from umbilical cord-derived MSCs using human fetal lung-derived fibroblasts and the mRNA expression level of the p21 gene (a cell proliferation-inhibiting gene) as an indicator. FIG. 8 shows the results of evaluating the cell aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from umbilical cord-derived MSCs using human fetal lung-derived fibroblasts and the mRNA expression level of the p53 gene (a cell proliferation-inhibiting gene) as an indicator. Figure 9 shows the results of evaluating the cellular aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from umbilical cord-derived MSCs using human fetal lung-derived fibroblasts and the mRNA expression level of the MMP-1 gene (collagen-degrading enzyme gene) as an indicator. FIG. 10 shows the primers used in quantitative PCR. FIG. 11 shows the results of evaluating the cellular aging-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs or (B) adipose-derived MSCs using human skin-derived fibroblasts (TIG3S cells) and the mRNA expression level of the p16 gene (a cell proliferation-inhibiting gene) as an indicator. FIG. 12 shows the results of evaluating the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs, (B) umbilical cord-derived MSCs, or (C) adipose-derived MSCs, using human skin-derived fibroblasts as an indicator of the mRNA expression level of the p21 gene (a cell proliferation-inhibiting gene). FIG. 13 shows the results of evaluating the cellular aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs, (B) umbilical cord-derived MSCs, or (C) adipose-derived MSCs, using human skin-derived fibroblasts as an indicator of mRNA expression level of the MMP-1 gene (collagen-degrading enzyme gene). FIG. 14 shows the results of evaluating the cellular senescence-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs, (B) umbilical cord-derived MSCs, or (C) adipose-derived MSCs, using human skin-derived fibroblasts as an indicator of the mRNA expression level of the COL1A1 gene (type I collagen gene). FIG. 15 shows the results of evaluating the cellular senescence-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs, (B) umbilical cord-derived MSCs, or (C) adipose-derived MSCs, using human skin-derived fibroblasts as an indicator of the mRNA expression level of the COL3A1 gene (type III collagen gene). FIG. 16 shows the results of evaluating the cellular aging-inhibiting activity and tissue repair-promoting activity of extracellular vesicles obtained from (A) bone marrow-derived MSCs, (B) umbilical cord-derived MSCs, or (C) adipose-derived MSCs, using human skin-derived fibroblasts as an indicator of the mRNA expression level of the HAS1 gene (hyaluronan synthase gene).

The basis of the present invention is a cellular aging inhibitor, a biological tissue repair promoter, and a gene expression regulator, which contain as active ingredients extracellular vesicles obtained by a method utilizing extracellular vesicles derived from mesenchymal stem cells stimulated with inflammatory cytokines and/or substances having affinity for phosphatidylserine, as described above.

Extracellular vesicles (hereinafter sometimes abbreviated as "EV") are small membrane vesicles derived from cells and composed of a lipid bilayer membrane. The extracellular vesicles generally have a diameter of 20 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 50 nm to 500 nm, and particularly preferably 50 nm to 200 nm. As described in Nature Reviews Immunology 9, 581-593 (August 2009) and "Obesity Research" Vol. 13 No. 2 2007 Topics Naoto Aoki et al., the extracellular vesicles are classified in various ways according to their origin and the size of the small membrane vesicles. Specific examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adiposomes, etc., with exosomes and microvesicles being preferred, and exosomes being more preferred.

The exosomes are small membrane vesicles derived from cells and composed of a lipid bilayer membrane, and examples of such vesicles include those having a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Exosomes are believed to be derived from late endosomes.

The microvesicles are small membrane vesicles composed of a lipid bilayer membrane derived from cells, and may have a diameter of, for example, 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. Microvesicles are believed to be derived from cell membranes.

MSCs are stem cells capable of differentiating into cells belonging to mesoderm-derived tissues (mesenchymal system), such as osteoblasts, adipocytes, muscle cells, and chondrocytes. MSCs can be isolated from fat, bone marrow, umbilical cord matrix, and the like, and the MSCs used in the present invention (hereinafter sometimes abbreviated as "MSCs according to the present invention") can be obtained, for example, by a method of isolating them from mesoderm-derived tissues or a method of inducing them from stem cells such as iPS cells and ES cells. As the MSCs according to the present invention, those derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane, and placenta, or those derived from iPS cells are preferably used. The MSCs according to the present invention may be pretreated by collection, concentration, purification, isolation, dilution with a buffer solution, filtration sterilization, etc. These pretreatments may be performed appropriately according to conventional methods. In addition, the MSCs according to the present invention are preferably stimulated with inflammatory cytokines. The inflammatory cytokine may be any cytokine capable of inducing inflammatory symptoms in the body, and examples thereof include tumor necrosis factor α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ, with tumor necrosis factor α (TNFα), interleukin 1, and interferon γ being preferred, tumor necrosis factor α and interleukin 1 being more preferred, and tumor necrosis factor α being particularly preferred. The stimulation by the inflammatory cytokine may be performed using at least one of the above-mentioned cytokines, and may also be performed using two or more cytokines.

<Extracellular vesicles according to the present invention>
The extracellular vesicles (hereinafter sometimes abbreviated as "extracellular vesicles of the present invention") which are active ingredients in the cellular aging inhibitor, biological tissue repair promoter, gene expression regulator, and topical skin composition provided by the present invention are extracellular vesicles derived from the MSCs of the present invention stimulated with inflammatory cytokines (hereinafter sometimes abbreviated as "extracellular vesicles derived from stimulated MSCs"), and/or extracellular vesicles obtained using a substance having affinity for phosphatidylserine (hereinafter sometimes abbreviated as "PS-positive extracellular vesicles").

Extracellular vesicles derived from stimulated MSCs are obtained by isolating them from the cell culture supernatant of the MSCs of the present invention, which contain EVs stimulated with the inflammatory cytokines.

The method for obtaining stimulated MSC-derived extracellular vesicles may be any conventional method for isolating EVs from a sample, such as affinity methods (e.g., PS affinity methods), fractional centrifugation methods (e.g., ultracentrifugation methods such as pellet down method, sucrose cushion method, and density gradient centrifugation), immunoprecipitation methods, chromatography methods (e.g., ion exchange chromatography method, gel permeation chromatography method), density gradient methods (e.g., sucrose density gradient method), electrophoresis methods (e.g., organelle electrophoresis), magnetic separation methods (e.g., magnetic activated cell sorting (MACS) method), ultrafiltration concentration methods (e.g., nanomembrane ultrafiltration concentration method), Percoll gradient isolation method, methods using microfluidic devices, PEG precipitation methods, etc., and the affinity method, which can obtain highly purified extracellular membrane vesicles, or the fractional centrifugation method, which theoretically allows for unbiased recovery, is preferred, and the affinity method or ultracentrifugation method is more preferred, with the affinity method being particularly preferred. Among affinity methods, the PS affinity method is preferred. The affinity method and the fractional centrifugation method may be carried out, for example, according to the method described in JP 2016-088689 A. These isolation methods may be used alone or in combination of two or more. Furthermore, isolation by one isolation method may be repeated two or more times.

The cell culture supernatant of the MSC according to the present invention, which contains EVs stimulated with the inflammatory cytokines, can be obtained, for example, by proliferating the MSC according to the present invention stimulated with the inflammatory cytokines by cell culture, and further culturing the proliferated cells in an EV production medium. The stimulation of the MSC according to the present invention with the inflammatory cytokines can be achieved by culturing the MSC according to the present invention in the presence of the inflammatory cytokines. The cell culture of the MSC according to the present invention and the culture in an EV production medium can be performed according to the conventional methods used in this field, and the medium and culture conditions used are not particularly limited.

PS-positive extracellular vesicles are PS-positive (containing PS) extracellular vesicles in which phosphatidylserine is thought to be exposed on the membrane surface of the extracellular vesicles.

The substance having affinity for phosphatidylserine (hereinafter sometimes abbreviated as "PS affinity substance") may be any substance capable of specifically binding to phosphatidylserine constituting the membrane of extracellular vesicles, and examples thereof include Annexin V; MFG-E8; Tim1 protein (T cell immunoglobulin mucin domain-containing molecule 1, T-cell immunoglobulin-mucin domain 1), Tim2 protein (T cell immunoglobulin mucin domain-containing molecule 2, T-cell immunoglobulin-mucin domain 2), Tim3 protein (T cell immunoglobulin mucin domain-containing molecule 3, T-cell immunoglobulin-mucin domain 3), and Tim4 protein (T cell immunoglobulin mucin domain-containing molecule 4, T-cell immunoglobulin-mucin domain 4). Examples of the Tim protein include Tim proteins such as immunoglobulin-mucin-domain 4), and Tim proteins are preferred because they enable efficient acquisition of extracellular vesicles. More preferred are those selected from Tim4 protein, Tim3 protein, and Tim1 protein, with Tim4 protein and Tim1 protein being even more preferred, and Tim4 protein being particularly preferred.

The PS-positive extracellular vesicles can be obtained by isolating them from the cell culture supernatant of the MSC according to the present invention containing EVs (hereinafter sometimes abbreviated as "cell culture supernatant containing EVs") using a substance having affinity for phosphatidylserine, or by obtaining EVs from the cell culture supernatant of the MSC according to the present invention containing EVs by a conventional method in this field, such as ultracentrifugation, and then isolating them from the obtained EVs using a substance having affinity for phosphatidylserine. Among these, it is preferable to directly isolate them from the cell culture supernatant of the MSC according to the present invention containing EVs using a substance having affinity for phosphatidylserine.
The cell culture supernatant containing the EVs can be obtained, for example, by growing the MSCs according to the present invention by cell culture and further culturing the grown cells in an EV production medium. The cell culture of the MSCs according to the present invention and the culture in an EV production medium may be performed according to a conventional method used in this field, and the medium and culture conditions used are not particularly limited.

The following is an overview of a method for obtaining extracellular vesicles using a substance that has affinity for phosphatidylserine (hereinafter, sometimes abbreviated as the "PS affinity method"). A specific example of the PS affinity method is described in Patent Document 1, for example.

The PS affinity method involves contacting a cell culture supernatant containing the EVs with a PS affinity substance in the presence of calcium ions to form a complex between the extracellular vesicles in the cell culture supernatant and the PS affinity substance (hereinafter sometimes abbreviated as the "complex of the present invention"), and then separating the PS affinity substance from the complex to obtain PS-positive extracellular vesicles.

A preferred method of the PS affinity method specifically includes the following steps:
(1) contacting a cell culture supernatant containing EVs with a PS affinity substance in the presence of calcium ions to form a complex (the complex according to the present invention) between PS-positive extracellular vesicles in the cell culture supernatant and the PS affinity substance (hereinafter, sometimes abbreviated as a "complex formation step");
(2) separating the complex according to the present invention obtained in the complex formation step from the cell culture supernatant containing the EV (hereinafter, sometimes abbreviated as "complex separation step");
(3) Separating PS-positive extracellular vesicles from the complex of the present invention and obtaining PS-positive extracellular vesicles (hereinafter sometimes abbreviated as "obtaining step").

The cell culture supernatant containing EVs and the PS affinity substance used in the PS affinity method are the same as those described above, and the preferred ones and specific examples are also the same.

It is preferable that the PS affinity substance used in the complex formation step is immobilized (bound) to an insoluble carrier. In this case, the complex according to the present invention can be separated from the cell culture supernatant containing the EVs by a known B/F separation method in the complex separation step.

An example of a method for immobilizing a PS affinity substance on an insoluble carrier is described below, but it can be obtained, for example, by the method described in Patent Document 1.

Examples of insoluble carriers for immobilizing PS affinity substances include insoluble carriers used in immunological assays. Specific examples include organic substances such as polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, and ethylene-maleic anhydride copolymers; inorganic substances such as glass, silicon oxide, diatoms, porous glass, ground glass, alumina, silica gel, and metal oxides; magnetic substances such as iron, cobalt, nickel, magnetite, and chromite; and those prepared using alloys of these magnetic substances as materials. In addition, examples of the form in which these carriers are used include particles (beads), microplates, tubes, and disk-shaped pieces. The insoluble carrier is preferably in the form of particles (beads), and the size of the particles is not particularly limited, but may be, for example, 10 nm to 100 μm, and preferably 100 nm to 10 μm.

Methods for binding the PS affinity substance to the insoluble carrier include known methods for binding proteins to carriers. For example, methods for binding by affinity binding, methods for binding by chemical binding (e.g., the methods described in Japanese Patent Publication No. 3269554 and WO2012/039395), and methods for binding by physical adsorption (e.g., the methods described in Japanese Patent Publication No. 5-41946) are included. The methods for binding by physical adsorption and the methods for binding by affinity binding are preferred, and the method for binding by physical adsorption is more preferred because it is simple.

A method for binding the PS affinity substance to the insoluble carrier by physical adsorption involves contacting the PS affinity substance with the insoluble carrier under conditions in which the PS affinity substance binds to the insoluble carrier, according to a method known per se.

The amount of PS affinity substance to be bound to the insoluble carrier may be, for example, 0.1 μg to 50 μg per 1 mg of insoluble carrier when the insoluble carrier is a particle (bead), preferably 0.1 μg to 30 μg, and more preferably 0.1 μg to 20 μg.

Physical adsorption between the PS affinity substance and the insoluble carrier can be carried out, for example, by contacting a solution containing the PS affinity substance with the insoluble carrier.

In the solution containing the PS affinity substance, the solution in which the PS affinity substance is dissolved may be any solution that dissolves the PS affinity substance in a stable state, such as purified water, or a buffer solution having a buffering effect at pH 6.0 to 9.8, preferably 7.0 to 9.6 (e.g., Good's buffer such as MOPS, carbonate buffer, PBS, TBS, TBS-T, HBS, etc.). The buffer concentration in these buffer solutions is usually selected appropriately from the range of 5 to 100 mM, preferably 10 to 100 mM. When NaCl is contained, the concentration is, for example, 100 to 200 mM, and preferably 140 to 160 mM. In addition, the solution containing the PS affinity substance may contain, for example, sugars, salts such as NaCl, surfactants such as Tween 20, preservatives, proteins, etc., as long as the amount does not interfere with the binding of the PS affinity substance to the insoluble carrier.

Specific examples of methods for binding the PS affinity substance to the insoluble carrier by physical adsorption include the following methods: For example, 1 mg of bead (particle) carrier is contacted with a solution containing 0.1 μg to 50 μg, preferably 0.1 μg to 30 μg, more preferably 0.1 μg to 20 μg of the PS affinity substance, and reacted at 2°C to 37°C, preferably 4°C to 11°C, for 0.5 to 48 hours, preferably 0.5 to 24 hours.

The insoluble carrier having the PS affinity substance immobilized thereon obtained as described above may be subjected to a blocking treatment which is typically performed in this field.

The complex formation step is carried out in the presence of calcium ions. The calcium ions are present when the PS affinity substance is contacted with the PS-positive extracellular vesicles in the cell culture supernatant containing the EVs. The calcium ion concentration when the PS affinity substance is contacted with the PS-positive extracellular vesicles in the cell culture supernatant containing the EVs is usually 0.5 mM to 100 mM, preferably 1.0 mM to 10 mM, more preferably 2.0 mM to 5.0 mM.
The above-mentioned concentration of calcium ions is required in the solution containing the complex of the present invention until the complex separation step is carried out after the complex of the present invention is formed between the PS affinity substance and the PS-positive extracellular vesicles in the cell culture supernatant containing the EVs, i.e., until the complex of the present invention is subjected to the step of separating the complex.

The origin of the calcium ion is not particularly limited, and examples include calcium chloride, calcium hydroxide, calcium bicarbonate, calcium iodide, calcium bromide, calcium acetate, etc., with calcium chloride, calcium bicarbonate, and calcium iodide being preferred, and calcium chloride and calcium bicarbonate being more preferred.

As a method for making calcium ions present when the PS affinity substance is brought into contact with the PS-positive extracellular vesicles in the cell culture supernatant containing the EV, the above-mentioned calcium ions may be contained in the cell culture supernatant containing the EV or/and a solution containing the PS affinity substance so that the calcium ion concentration is within the above-mentioned range when the PS affinity substance is brought into contact with the PS-positive extracellular vesicles in the cell culture supernatant containing the EV. In addition, a solution containing calcium ions in an amount that makes the calcium ion concentration within the above-mentioned range when the PS affinity substance is brought into contact with the PS-positive extracellular vesicles in the cell culture supernatant containing the EV (hereinafter sometimes abbreviated as the "calcium ion-containing solution according to the present invention") may be mixed with the cell culture supernatant containing the EV and a solution containing the PS affinity substance.

In the calcium ion-containing solution according to the present invention, the solution in which calcium ions are dissolved may be any solution that does not interfere with the binding of PS-positive extracellular vesicles to PS affinity substances, and examples of such solutions include water and buffers having a buffering effect at pH 7.0 to pH 8.0, and preferably buffers having a buffering effect at pH 7.2 to pH 7.6 (e.g., TBS, HBS, etc.). Phosphate buffers are not preferred because they bind to calcium and cause precipitation. The buffer concentration in these buffers is usually selected from the range of 5 mM to 50 mM, preferably 10 mM to 30 mM. When NaCl is contained, the concentration is usually selected from the range of 100 mM to 200 mM, preferably 140 mM to 160 mM.

The calcium ion-containing solution according to the present invention may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins such as BSA, etc., in amounts that do not interfere with the binding of PS-positive extracellular vesicles to the PS affinity substance. Examples of surfactants include Tween 20, and the concentration of the surfactant in the calcium ion-containing solution according to the present invention is usually 0.00001% to 0.2%, preferably usually 0.0005% to 0.1%.

In the complex formation step, the amount of the cell culture supernatant containing the EVs to be contacted with 1 μg of the PS affinity substance (which may be immobilized on the insoluble carrier) is usually 0.1 ml to 100 ml, preferably 0.1 ml to 10 ml, and more preferably 0.1 ml to 1.0 ml. The temperature at which the cell culture supernatant containing the EVs is contacted with the PS affinity substance is usually 2 to 37° C., preferably 4 to 37° C., and more preferably 4 to 30° C. The contact time between the cell culture supernatant containing the EVs and the PS affinity substance is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

In the complex formation process, when an insoluble carrier to which a PS affinity substance is immobilized is used, the amount of the carrier is typically 0.1 mg to 20 mg per mL of solution used to form the complex of the present invention, preferably 0.3 mg to 10 mg, and more preferably 0.5 mg to 6.0 mg.

The complex formation step may be carried out, for example, by the following method. That is, the insoluble carrier having the PS affinity substance immobilized thereon is usually 0.1 mg to 20 mg, preferably 0.3 mg to 10 mg, and more preferably 0.5 mg to 6.0 mg per mL of the solution obtained after mixing the cell culture supernatant containing the EV, the insoluble carrier having the PS affinity substance immobilized thereon, and the calcium ion-containing solution according to the present invention, and the calcium ion concentration in the solution obtained after mixing the cell culture supernatant containing the EV, the carrier, and the calcium ion-containing solution according to the present invention is usually 0.5 mM to 100 mM, preferably 1.0 mM to 10 mM, and more preferably 2.0 mM to 5.0 mM. The calcium ion-containing solution according to the present invention is contacted with a cell culture supernatant containing the EVs in an amount of usually 0.1 ml to 100 ml, preferably 0.1 ml to 10 ml, and more preferably 0.1 ml to 1.0 ml per 1 mg of an insoluble carrier having a PS affinity substance immobilized thereon, usually at 4.0 to 37° C., preferably 4.0 to 25° C., and more preferably 4.0 to 11° C., usually for 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and more preferably 0.5 to 4.0 hours, to form a complex (the complex according to the present invention) between the PS affinity substance bound to the carrier and the PS-positive extracellular vesicles in the cell culture supernatant containing the EVs.

The complex separation step may be any method that can separate the complex of the present invention from the cell culture supernatant containing the EV to obtain the complex of the present invention, but examples of the method include the following:

(1) When the insoluble carrier to which the PS affinity substance is immobilized is a magnetic carrier: A method in which a container containing the complex of the present invention obtained by the complex formation process is placed on a magnetic stand, if necessary, and the complex of the present invention is aggregated on the tube wall using magnetic force, and the supernatant is removed to separate them. (2) When the insoluble carrier to which the PS affinity substance is immobilized is in the form of beads: A method in which a container containing the complex of the present invention obtained by the complex formation process is centrifuged to aggregate the complex of the present invention as a precipitate, and the supernatant is removed to separate them. (3) A method in which the complex of the present invention and the cell culture supernatant containing the EV are separated by filtration.

Specific examples of the complex separation step include the following methods.
When a magnetic carrier is used as the insoluble carrier, the container in which the complex formation step has been carried out is placed on a magnetic stand, if necessary, and the complex according to the present invention obtained is collected on the tube wall using magnetic force, and the supernatant sample is removed.

After the complex separation step, if necessary, the complex according to the present invention obtained may be washed with a washing solution containing calcium ions (hereinafter, sometimes abbreviated as "washing operation"). The washing operation can remove impurities in the biological sample, such as cell-derived components attached to the surface of the insoluble carrier on which the PS affinity substance is immobilized. As for the washing method, any washing method usually used in this field can be used, except for using a washing solution containing calcium ions.

The calcium ion-containing washing solution used in the washing operation may be any solution that contains calcium ions at 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, and does not affect the binding between PS-positive extracellular vesicles and the PS affinity substance immobilized on the insoluble carrier. For example, a buffer solution that does not precipitate calcium and has a buffering effect at pH 7.0 to pH 8.0, preferably pH 7.2 to pH 7.6, containing calcium ions at 0.5 mM to 100 mM, preferably 1 mM to 10 mM, more preferably 2 mM to 5 mM (e.g., TBS, TBS-T, HBS). Phosphate buffer is not preferred because it binds to calcium and causes precipitation. The buffer concentration in these buffer solutions is usually selected from the range of 5 mM to 50 mM, preferably 10 mM to 30 mM, and the concentration when NaCl is contained is usually selected from the range of 100 mM to 200 mM, preferably 140 mM to 160 mM. This solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., in amounts that do not interfere with the binding between the PS-positive extracellular vesicles and the PS affinity substance immobilized on the insoluble carrier. Examples of surfactants include Tween 20 (FUJIFILM Wako Pure Chemical Industries, Ltd.), and the concentration of the surfactant in the washing solution is usually 0.00001% to 0.2%, preferably usually 0.0005% to 0.1%.

A specific example of the washing operation will be described below using as an example a washing operation using magnetic particles as an insoluble carrier for immobilizing the PS affinity substance. That is, a washing solution containing calcium ions according to the present invention is added to a container containing the complex according to the present invention obtained by the complex separation process, and stirred. The container is then placed on a magnetic stand, the complex according to the present invention is aggregated on the tube wall using magnetic force, and the solution in the container is discarded. These washing operations may be repeated several times as necessary.

The acquisition step may be any method capable of acquiring PS-positive extracellular vesicles from the complex of the present invention, and a method of reducing the calcium ion concentration is preferred. An example of a method of reducing the calcium ion concentration is a method using a calcium ion chelating agent. That is, after the complex separation step, and if necessary after a washing operation, a calcium ion chelating agent is applied to calcium ions in the reaction system (calcium ions bound to the complex of the present invention and calcium ions brought in from the solution containing the complex of the present invention) to chelate the calcium ions, thereby reducing the effective concentration of calcium ions in the reaction system, thereby separating PS-positive extracellular vesicles from the complex of the present invention.

The calcium ion chelating agent used in this method may be any compound capable of chelating calcium ions, such as EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), GLDA (L-glutamic acid diacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), GEDTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N,N,-tetraacetic acid), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), HIDA (2-hydroxyethyliminodiacetic acid), DHEG (N,N-bis(2-hydroxyethyl)glycine), CyDTA (trans-1z,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid), Of these, EDTA, GEDTA, and CyDTA are preferred.

The calcium ion chelating agent is usually used as a solution. The solution in which the calcium ion chelating agent is dissolved may be any solution that dissolves the calcium ion chelating agent, and examples thereof include purified water and buffer solutions. As the buffer solution, a buffer solution having a buffering effect at pH 7.0 to pH 8.0, preferably pH 7.2 to pH 7.6 (e.g., PBS, TBS, HBS, etc.) is usually preferred. The buffer concentration in these buffer solutions is usually appropriately selected from the range of 5 Mm to 50 Mm, preferably 10 Mm to 30 Mm, and the concentration when NaCl is contained is usually appropriately selected from the range of 100 Mm to 200 Mm, preferably 140 Mm to 160 Mm. The solution containing the calcium ion chelating agent (hereinafter sometimes abbreviated as "calcium ion chelating agent-containing solution") may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

The concentration of the calcium ion chelating agent in the calcium ion chelating agent-containing solution is usually 0.5 mM to 500 mM, preferably 0.5 mM to 100 mM, and more preferably 0.5 mM to 50 mM. The pH of the calcium ion chelating agent-containing solution is usually pH 6.0 to pH 9.0, preferably pH 7.0 to pH 8.0, and more preferably pH 7.2 to pH 7.6.

To allow the calcium ion chelating agent to act on the calcium ions bound to the complex of the present invention, a solution containing the calcium ion chelating agent is brought into contact with the complex of the present invention (e.g., in pellet form) and the calcium ions bound to the complex of the present invention are reacted with the calcium ion chelating agent in the calcium ion chelating agent-containing solution.

Contact between the calcium ion chelating agent-containing solution and the complex of the present invention can be carried out, for example, by suspending the complex of the present invention in the calcium ion chelating agent-containing solution (when the insoluble carrier to which the PS affinity substance is immobilized is a bead, etc.), or by immersing the complex of the present invention in the calcium ion chelating agent-containing solution (when the insoluble carrier to which the PS affinity substance is immobilized is a disk-shaped piece or a tube, etc.).

The amount of calcium ion chelating agent-containing solution to be contacted with the complex of the present invention should be an amount such that the concentration of calcium ions in the solution after contact with the complex of the present invention is less than the effective concentration and extracellular vesicles are separated from the complex of the present invention.

The temperature and time for allowing the calcium ion chelating agent to act (contact) on the complex of the present invention are typically 4.0°C to 37°C, preferably 10°C to 30°C, and more preferably 20°C to 30°C, and are typically 1 to 10 minutes, and preferably 5 to 15 minutes.

The acquisition step is described below by taking as an example a method using a carrier in which Tim protein is bound to an insoluble carrier (Tim carrier). That is, after the complex separation step and, if necessary, after a further washing operation, a solution containing usually 0.5 mM to 500 mM, preferably 0.5 mM to 100 mM, and more preferably 0.5 mM to 50 mM calcium ion chelating agent is added to the obtained complex of the present invention in an amount of usually 10 μL to 500 μL, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL per 1 mg of Tim carrier, and reacted at usually 4.0° C. to 37° C., preferably 10° C. to 30° C., and more preferably 20° C. to 30° C., for usually 1 to 30 minutes, preferably 5 to 15 minutes, to separate PS-positive extracellular vesicles from the complex of the present invention.

By carrying out the obtaining step, the calcium ion chelating agent-containing solution that has been brought into contact with the complex of the present invention contains the insoluble carrier on which the PS affinity substance is immobilized and the extracellular vesicles that have been separated (released) from the complex of the present invention. Therefore, by removing the carrier on which the PS affinity substance is immobilized from the solution and recovering only the solution, a solution containing PS-positive extracellular vesicles can be obtained.

The extracellular vesicles according to the present invention are preferably stimulated MSC-derived extracellular vesicles, more preferably derived from mesenchymal stem cells stimulated with the inflammatory cytokine, and obtained by a method using a substance having affinity for phosphatidylserine, and further preferably derived from mesenchymal stem cells stimulated with at least one or more inflammatory cytokines selected from tumor necrosis factor α (TNFα), interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 18, and interferon γ, and obtained by a PS affinity method using Tim protein, and further preferably derived from mesenchymal stem cells stimulated with at least one or more inflammatory cytokines selected from tumor necrosis factor α (TNFα), interleukin 18, and interferon γ, and obtained by a PS affinity method using Tim protein, and further preferably derived from mesenchymal stem cells stimulated with at least one or more inflammatory cytokines selected from tumor necrosis factor α, ... More preferably, the protease is derived from mesenchymal stem cells stimulated with at least one inflammatory cytokine selected from interleukin 1 and interferon gamma and obtained by the PS affinity method using Tim4 protein, Tim3 protein or Tim1 protein; particularly preferably, the protease is derived from mesenchymal stem cells stimulated with tumor necrosis factor α or interleukin 1 and obtained by the PS affinity method using Tim4 protein; and most preferably, the protease is derived from mesenchymal stem cells stimulated with tumor necrosis factor α and obtained by the PS affinity method using Tim4 protein.

<Cellular aging inhibitor of the present invention>
The cellular aging inhibitor of the present invention contains the extracellular vesicles of the present invention as an active ingredient. The cellular aging inhibitor of the present invention can inhibit cellular aging by at least one cellular aging inhibitory effect selected from the following: cell proliferation promoting effect, collagen production promoting effect or collagen degradation inhibiting effect, hyaluronic acid production promoting effect or collagen degradation inhibiting effect, and elastin production promoting effect or collagen degradation inhibiting effect, which are caused by regulating the expression of cellular aging-related genes.

The cell aging-related gene may be any gene related to cell aging, for example, cell proliferation inhibitory genes such as the p53 gene, p21 gene, and p16 gene; collagen genes such as type I collagen genes (COL1A1 gene, COL1A2 gene), type III collagen genes (COL3A1 gene), type V collagen genes (COL5A1 gene, COL5A2 gene), and type XVII collagen gene (COL17A1 gene); collagen decomposition enzyme genes such as the MMP-1 gene, MMP-2 gene, MMP-3 gene, MMP-8 gene, MMP-9 gene, and MMP-13 gene; Examples of the hyaluronic acid synthase genes include hyaluronic acid synthase 1 gene (HAS1 gene) and hyaluronic acid synthase 2 gene (HAS2 gene); hyaluronic acid decomposition enzyme genes such as HYAL1 gene, HYAL2 gene, and HYAL3 gene; elastin genes such as ELN gene; and elastin decomposition enzyme genes such as MMP-2 gene and MMP-9 gene. Cell proliferation inhibitory genes, collagen genes, collagen decomposition enzyme genes, and hyaluronic acid synthase are preferred, and p53 gene, p21 gene, p16 gene, MMP-1 gene, COL1A1 gene, COL3A1 gene, and HAS1 gene are more preferred. The cell proliferation inhibitory gene may be any gene involved in the inhibition of cell proliferation, and examples thereof include genes encoding cell proliferation inhibitory factors. It is believed that cyclin kinase involved in cell division is inhibited by regulating the expression of genes encoding cell proliferation inhibitory factors, thereby inhibiting cell proliferation.

In the present invention, regulation of gene expression of a cellular senescence-related gene means promotion and/or inhibition of expression of the cellular senescence-related gene, and specific examples include promotion of collagen gene expression, promotion of hyaluronic acid synthase gene expression, promotion of elastin gene expression, inhibition of cell proliferation-inhibiting gene expression, inhibition of collagen decomposition enzyme gene expression, inhibition of hyaluronic acid decomposition enzyme gene expression, and inhibition of elastin decomposition enzyme gene expression. Promotion of collagen gene expression, promotion of hyaluronic acid synthase gene expression, inhibition of cell proliferation-inhibiting gene expression, and inhibition of collagen decomposition enzyme gene expression are preferred, and promotion of COL1A1 gene expression, promotion of COL3A1 gene expression, promotion of HAS1 gene expression, inhibition of p53 gene expression, inhibition of p21 gene expression, inhibition of p16 gene expression, and inhibition of MMP-1 gene expression are more preferred.

The cells whose cellular senescence is inhibited by the cellular senescence inhibitor of the present invention are not particularly limited, and examples thereof include fibroblasts, with dermal fibroblasts being more preferred.

The content of the extracellular vesicles of the present invention in the cellular senescence agent of the present invention is not particularly limited as long as the cellular senescence inhibitory activity (cellular senescence inhibitory effect) of the extracellular vesicles of the present invention is exhibited. Furthermore, there are no limitations on the form of the cellular senescence agent of the present invention or on the presence or absence of components other than the extracellular vesicles of the present invention. Furthermore, the cellular senescence inhibitory activity in the present invention can be evaluated using the expression level of the cellular senescence-related gene as an index.

The cellular aging inhibitor of the present invention contains the extracellular vesicles of the present invention as an active ingredient, and is provided, for example, as a pharmaceutical composition. The dosage form of the cellular aging inhibitor of the present invention may be a solution containing the extracellular vesicles of the present invention as is, or may be formulated as a liquid, suspension, lipo-formation agent, etc. together with a pharma-ceutically acceptable carrier or additive as necessary, or may be freeze-dried to form a powder, or may be formulated as a solid preparation such as a tablet together with a pharma-ceutically acceptable additive.

Examples of pharma- ceutically acceptable carriers and additives used in the formulation include, but are not limited to, isotonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersing agents, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, acidulants, colorants, flavoring agents, fragrances, or cooling agents.

Representative examples of carriers, additives, etc. include, for example, the following. Carriers include, for example, aqueous carriers such as water and aqueous ethanol. Isotonicity agents include, for example, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. Polyhydric alcohols include, for example, glycerin, propylene glycol, and polyethylene glycol. Thickeners include, for example, carboxyvinyl polymer, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, alginic acid, polyvinyl alcohol (completely or partially saponified), polyvinylpyrrolidone, and macrogol.

Examples of sugars include cyclodextrin, glucose, fructose, lactose, etc. Examples of sugar alcohols include sugar alcohols such as xylitol, sorbitol, mannitol, etc. Examples of preservatives, disinfectants, or antibacterial agents include dibutylhydroxytoluene, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate. Examples of pH adjusters include hydrochloric acid, boric acid, aminoethylsulfonic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, magnesium sulfate, phosphoric acid, polyphosphoric acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, and succinic acid.

Examples of stabilizers include dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (Rongalit), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glycerin monostearate, sodium hydrogensulfite, sodium sulfite, etc. Examples of bases include vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, cottonseed oil, etc.; oily bases such as medium-chain fatty acid triglycerides; aqueous bases such as Macrogol 400; gel bases such as carboxyvinyl polymers and gums. Examples of surfactants include polysorbate 80, hydrogenated castor oil, glycerin fatty acid esters, sorbitan sesquioleate, etc. Examples of suspending agents include white beeswax, various surfactants, gum arabic, powdered gum arabic, xanthan gum, soy lecithin, etc.

Furthermore, examples of binders include hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc., examples of excipients include sucrose, lactose, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc., examples of lubricants include sucrose fatty acid esters, magnesium stearate, talc, etc., examples of disintegrants include low-substituted hydroxypropyl cellulose, crospovidone, croscarmellose sodium, etc., and examples of flow agents include sodium aluminometasilicate, light anhydrous silicic acid, etc.

The cell aging inhibitor of the present invention is preferably formulated as a liquid, suspension, or lipo-formulated agent, and is basically obtained by mixing a solution containing the extracellular vesicles of the present invention with the above-mentioned carriers and additives as necessary, for example, physiological saline, 5% glucose solution, lipo-emulsion, etc. It is also possible to use a lyophilized powder and prepare a preparation that is dissolved or suspended when used.

When the cell aging inhibitor of the present invention is a liquid, suspension or liposomal agent, its pH is not particularly limited as long as it is within a medicamentously, pharmacologically or physiologically acceptable range, but examples of such ranges include a pH range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0, and can be appropriately adjusted with a pH adjuster.

The administration route of the cell aging inhibitor of the present invention may be oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, or intraperitoneal administration, depending on the dosage form. The dosage varies depending on the condition of the subject patient (body weight, age, symptoms, physical condition, etc.) and the dosage form, etc., but usually, when administered to an adult, the particle number is 1×10 ⁵ to 1×10 ¹⁷ particles/time, preferably 5×10 ⁵ to 5×10 ¹⁶ particles/time, more preferably 1×10 ⁶ to 1×10 ¹⁶ particles/time, and particularly preferably 5×10 ⁶ to 5×10 ¹⁵ particles/time. This dosage may be administered multiple times a day as a single dosage, or this dosage may be administered in multiple divided doses.

The cellular aging inhibitor of the present invention may be formulated as an external preparation for the skin, such as a transdermal drug or a transdermal quasi-drug. The content of the extracellular vesicles of the present invention in the external preparation for the skin is not particularly limited as long as the cellular aging inhibitory activity of the extracellular vesicles of the present invention is exhibited, and is, for example, preferably 1x10 ⁶ particles/mL to 1x10 ¹⁵ particles/mL, more preferably 1x10 ⁷ particles/mL to 1x10 ¹⁴ particles/mL, and particularly preferably 1x10 ⁸ particles/mL to 1x10 ¹³ particles/mL.

The skin topical preparation may be in any dosage form, for example, a solubilized system such as lotion, a dispersion system such as calamine lotion, or an emulsion system such as cream or milky lotion. It may also be in various dosage forms, such as an aerosol filled with a propellant, a pump spray, an ointment, a cataplasm, a tape, or an injection. In addition to the extracellular vesicles according to the present invention, the skin topical preparation may contain any component (base material) that is usually blended in transdermal pharmaceuticals, transdermal quasi-drugs, etc., depending on the purpose and necessity. Examples of such components include water, oily components, moisturizers, powders, pigments, emulsifiers, solubilizers, gelling agents, detergents, ultraviolet absorbers, anti-inflammatory agents, antiallergic agents, thickeners, pH adjusters, chelating agents, medicines (medicinal components), fragrances, resins, antibacterial and antifungal agents, preservatives (preservatives), antioxidants, slimming agents, alcohols, surfactants, ultraviolet absorbers, moisturizers, emollients, vitamins, colorants, natural extracts, etc. Furthermore, other compounds having a cellular aging inhibitory effect can be appropriately blended within a range that does not impair the effects of the present invention. The content of these optional components is not particularly limited, and can be appropriately selected depending on the desired dosage form, application, etc.

<Bio-tissue repair promoter of the present invention>
The biological tissue repair promoter of the present invention comprises the extracellular vesicles of the present invention as an active ingredient. According to the biological tissue repair promoter of the present invention, the expression of biological tissue repair-related genes is regulated, cells grow, and the repair of fibers constituting biological tissues such as collagen fibers and elastic fibers is promoted, thereby promoting the repair of biological tissues whose homeostatic functions have decreased or disappeared. Specifically, the repair of biological tissues can be promoted based on at least one biological tissue repair promoting action selected from the cell proliferation promoting action by regulating the gene expression of cell proliferation inhibitory genes, the collagen fiber (collagen fiber) repair promoting action by collagen production promoting action or collagen decomposition inhibiting action, the elastic fiber (elastin fiber) repair action by elastin production promoting action or elastin decomposition inhibiting action, and the hyaluronic acid production promoting action or hyaluronic acid decomposition inhibiting action.

The biological tissue repair-related gene may be any biological tissue repair-related gene, for example, cell proliferation inhibitory genes such as the p53 gene, p21 gene, and p16 gene; collagen genes such as type I collagen genes (COL1A1 gene, COL1A2 gene), type III collagen genes (COL3A1 gene), type V collagen genes (COL5A1 gene, COL5A2 gene), and type XVII collagen gene (COL17A1 gene); collagen decomposition enzyme genes such as the MMP-1 gene, MMP-2 gene, MMP-3 gene, MMP-8 gene, MMP-9 gene, and MMP-13 gene; Examples of the hyaluronic acid synthase gene include hyaluronic acid synthase 1 gene (HAS1 gene) and hyaluronic acid synthase 2 gene (HAS2 gene); hyaluronic acid decomposition enzyme gene such as HYAL1 gene, HYAL2 gene, and HYAL3 gene; elastin gene such as ELN gene; and elastin decomposition enzyme gene such as MMP-2 gene and MMP-9 gene. Cell proliferation inhibitory gene, collagen gene, collagen decomposition enzyme gene, and hyaluronic acid synthase are preferred, and p53 gene, p21 gene, p16 gene, MMP-1 gene, COL1A1 gene, COL3A1 gene, and HAS1 gene are more preferred. The cell proliferation inhibitory gene may be any gene involved in the inhibition of cell proliferation, and examples thereof include genes encoding cell proliferation inhibitory factors. It is believed that cyclin kinase involved in cell division is inhibited by regulating the expression of the gene encoding the cell proliferation inhibitory factor, thereby inhibiting cell proliferation.

In the present invention, regulating the expression of biological tissue repair-related genes means promoting and/or suppressing the expression of the biological tissue repair-related genes, and specific examples include promoting the expression of collagen genes, promoting the expression of hyaluronic acid synthase genes, promoting the expression of elastin genes, suppressing the expression of cell proliferation-inhibiting genes, suppressing the expression of collagen decomposition enzyme genes, suppressing the expression of hyaluronic acid decomposition enzyme genes, and suppressing the expression of elastin decomposition enzyme genes. Promotion of collagen gene expression, promotion of hyaluronic acid synthase gene expression, suppression of cell proliferation-inhibiting genes, and suppression of collagen decomposition enzyme gene expression are preferred, and promotion of COL1A1 gene expression, promotion of COL3A1 gene expression, promotion of HAS1 gene expression, suppression of p53 gene expression, suppression of p21 gene expression, suppression of p16 gene expression, and suppression of MMP-1 gene expression are more preferred.

Tissues whose repair is promoted by the biological tissue repair promoter of the present invention include tissues containing collagen fibers or elastic fibers and tissues containing hyaluronic acid, specifically, for example, the dermis, ligaments, connective tissues, arteries, lungs, etc., with the dermis and ligaments being preferred.

The content of the extracellular vesicles of the present invention in the biological tissue repair promoter of the present invention is not particularly limited as long as the biological tissue repair promoting activity (biological tissue repair promoting effect) of the extracellular vesicles of the present invention is exhibited. Furthermore, there are no limitations on the form of the biological tissue repair promoter of the present invention or on the presence or absence of components other than the extracellular vesicles of the present invention. Furthermore, the biological tissue repair promoting activity in the present invention can be evaluated using the expression level of the biological tissue repair-related gene as an index.

The biological tissue repair promoter of the present invention contains the extracellular vesicles of the present invention as an active ingredient, and is provided, for example, as a pharmaceutical composition. The dosage form of the biological tissue repair promoter of the present invention is a solution containing the extracellular vesicles of the present invention as is, or, if necessary, formulated as a liquid, suspension, lipo-formation agent, etc. together with a pharma-ceutically acceptable carrier or additive, or further, formulated as a powder by lyophilization, or as a solid preparation such as a tablet together with a pharma-ceutically acceptable additive.

The pharma- ceutically acceptable carriers and additives used in the formulation include the same as those of the cell aging inhibitor of the present invention described above, and the preferred ones are the same. The biological tissue repair promoter of the present invention is preferably formulated as a liquid, suspension, or lipo-formaldehyde agent, and is basically obtained by mixing a solution containing the extracellular vesicles of the present invention with the above-mentioned carriers and additives as necessary, for example, physiological saline, 5% glucose solution, lipo-emulsion, etc. It is also possible to use a lyophilized powder to prepare a formulation that is dissolved or suspended when used.

When the biological tissue repair promoter of the present invention is a liquid, suspension or liposomal agent, its pH is not particularly limited as long as it is within a medicamentarily, pharmacologically or physiologically acceptable range, but examples of such ranges include a pH range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0, and can be appropriately adjusted with a pH adjuster.

The administration route of the biological tissue repair promoter of the present invention may be oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, or intraperitoneal administration, depending on the dosage form. The dosage varies depending on the condition of the target patient (body weight, age, symptoms, physical condition, etc.) and the dosage form, etc., but usually, when administered to an adult, the particle number is 1 x 10 ⁵ to 1 x 10 ¹⁷ particles/time, preferably 5 x 10 ⁵ to 5 x 10 ¹⁶ particles/time, more preferably 1 x 10 ⁶ to 1 x 10 ¹⁶ particles/time, and particularly preferably 5 x 10 ⁶ to 5 x 10 ¹⁵ particles/time. This dosage may be administered multiple times a day as a single dosage, or this dosage may be administered in multiple doses.

The biological tissue repair promoter of the present invention may be formulated as an external preparation for skin such as a transdermal drug or a transdermal quasi-drug. The content of the extracellular vesicles of the present invention in the external preparation for skin is not particularly limited as long as the biological tissue repair promoting activity of the extracellular vesicles of the present invention is exhibited, and is, for example, preferably 1x10 ⁶ particles/mL to 1x10 ¹⁵ particles/mL, more preferably 1x10 ⁷ particles/mL to 1x10 ¹⁴ particles/mL, and particularly preferably 1x10 ⁸ particles/mL to 1x10 ¹³ particles/mL.

The dosage form of the skin topical preparation is arbitrary, and is the same as that of the skin topical preparation that provides the cell aging inhibitor of the present invention described above as a skin topical preparation. In addition to the extracellular vesicles of the present invention, the skin topical preparation may contain any component (base material) that is usually blended in transdermal pharmaceuticals, transdermal quasi-drugs, etc., depending on the application and necessity, and is the same as that of the skin topical preparation that provides the cell aging inhibitor of the present invention described above as a skin topical preparation. Furthermore, other compounds having a biological tissue repair promoting effect can be appropriately blended within a range that does not impair the effect of the present invention. In addition, the content of these optional components is not particularly limited, and can be appropriately selected according to the desired dosage form, application, etc.

<Composition for external use on skin of the present invention>
The topical skin composition for inhibiting cellular aging or promoting biological tissue repair of the present invention (hereinafter, sometimes abbreviated as "topical skin composition of the present invention") refers to any topical composition that contains the extracellular vesicles of the present invention as an active ingredient and is applied topically to the skin. Specifically, the topical skin composition of the present invention can be used for applications such as cosmetics, transdermal pharmaceuticals, and transdermal quasi-drugs.

The content of the extracellular vesicles according to the present invention in the composition for external use on the skin of the present invention is not particularly limited, as long as the extracellular vesicles according to the present invention exert their cellular aging inhibitory activity (cellular aging inhibitory effect) or biological tissue repair promoting activity (biological tissue repair promoting effect). The content of the extracellular vesicles according to the present invention in the composition for external use on the skin of the present invention is, for example, preferably 1x10 ⁶ particles/mL to 1x10 ¹⁵ particles/mL, more preferably 1x10 ⁷ particles/mL to 1x10 ¹⁴ particles/mL, and particularly preferably 1x10 ⁸ particles/mL to 1x10 ¹³ particles/mL.

The topical skin composition of the present invention may be provided in any dosage form, for example, as a solubilized system such as a lotion, a dispersion system such as calamine lotion, or an emulsion system such as a cream or milky lotion. Furthermore, it may be provided in various dosage forms, such as an aerosol filled with a propellant, a pump spray, an ointment, a cataplasm, a tape, or an injection.

Specific examples include various types of cosmetics such as emulsions, creams, lotions, skin toners, packs, beauty serums, cleansers, gels, and makeup cosmetics; cosmetics in various forms such as liquids, ointments, powders, granules, aerosols, pump sprays, patches, poultices, and tapes; transdermal pharmaceuticals; and transdermal quasi-drugs.

In addition to the extracellular vesicles according to the present invention, the skin topical composition of the present invention may contain any component (base material) that is usually blended in cosmetics such as skin cosmetics, transdermal medicines, transdermal quasi-drugs, and cleansers, etc., depending on the purpose and necessity. For example, water, oily components, moisturizers, powders, pigments, emulsifiers, solubilizers, gelling agents, cleansers, UV absorbers, anti-inflammatory agents, antiallergic agents, thickeners, pH adjusters, chelating agents, medicines (medicinal components), fragrances, resins, antibacterial and antifungal agents, preservatives (preservatives), antioxidants, slimming agents, alcohols, surfactants, UV absorbers, whitening agents, moisturizers, emollients, vitamins, colorants, natural extracts, etc. Furthermore, other compounds having a cell aging inhibitory effect or a compound having a biological tissue repair promoting effect can be appropriately blended within a range that does not impair the effects of the present invention. In addition, the content of these optional components is not particularly limited and can be appropriately selected according to the desired dosage form, purpose, etc.

The cells whose cellular senescence is inhibited by the topical skin composition of the present invention are similar to the cells whose cellular senescence is inhibited by the cellular senescence inhibitor of the present invention or the tissues whose repair is promoted by the biological tissue repair promoter of the present invention, and the same is also preferred.

The topical skin composition of the present invention may be produced in accordance with the method for producing the extracellular vesicles of the present invention or the cell aging inhibitor or biological tissue repair promoter of the present invention, and specific examples and preferred methods are also the same.

The skin topical composition of the present invention exhibits cellular aging inhibitory activity or biological tissue repair promoting activity by containing the extracellular vesicles of the present invention. When the skin topical composition of the present invention is applied to, for example, the facial skin or scalp, the expression of at least one of the cell aging-related genes or biological tissue repair-related genes is regulated, and based on the cell proliferation promoting action, collagen production promoting or decomposition inhibiting action, hyaluronic acid production promoting or decomposition inhibiting action, elastin production promoting or decomposition inhibiting action, etc., it can be expected to improve wrinkles and sagging caused by aging, treat or prevent hair loss or thinning, etc., or regenerate the function of cells, biological tissues, and organs that have become dysfunctional or dysfunctional.

<Gene expression regulator of the present invention>
The gene expression regulator of the present invention contains the extracellular vesicles of the present invention as an active ingredient, and is capable of suppressing cell proliferation such as p53 gene, p21 gene, and p16 gene; collagen genes such as type I collagen gene (COL1A1 gene, COL1A2 gene), type III collagen gene (COL3A1 gene), type V collagen gene (COL5A1 gene, COL5A2 gene), and type XVII collagen gene (COL17A1 gene); hyaluronan synthase 1 gene (HAS1 gene), hyaluronan synthase 2 gene (HAS2 gene), and The present invention is applied to the use of regulating the expression of at least one gene (hereinafter sometimes abbreviated as "genes to be expression-regulated") selected from hyaluronic acid synthase genes such as S2 gene, elastin genes such as ELN gene, collagen decomposition enzyme genes such as MMP-1 gene, MMP-2 gene, MMP-3 gene, MMP-8 gene, MMP-9 gene, MMP-13 gene, hyaluronic acid decomposition enzyme genes such as HYAL1 gene, HYAL2 gene, HYAL3 gene, and elastin decomposition enzyme genes such as MMP-2 gene, MMP-9 gene, etc. As the genes to be expression-regulated, cell proliferation inhibitory genes, collagen genes, collagen decomposition enzyme genes, and hyaluronic acid synthase genes are preferred, and p53 gene, p21 gene, p16 gene, MMP-1 gene, COL1A1 gene, COL3A1 gene, and HAS1 gene are more preferred.

In addition, gene expression regulation by the gene expression regulator of the present invention means promotion and/or inhibition of expression of the gene whose expression is to be regulated, and examples of such regulation include promotion of collagen gene expression, promotion of hyaluronic acid synthase gene expression, promotion of elastin gene expression, inhibition of cell proliferation inhibitory gene expression, inhibition of collagen decomposition enzyme gene expression, inhibition of hyaluronic acid decomposition enzyme gene expression, and inhibition of elastin decomposition enzyme gene expression. Promotion of collagen gene expression, promotion of hyaluronic acid synthase gene expression, inhibition of cell proliferation inhibitory gene expression, and inhibition of collagen decomposition enzyme gene expression are preferred, and inhibition of p53 gene expression, p21 gene expression, p16 gene expression, MMP-1 gene expression, promotion of COL1A1 gene expression, promotion of COL3A1 gene expression, and promotion of HAS1 gene expression are more preferred.

The cells whose gene expression is regulated by the gene expression regulator of the present invention are not particularly limited, and examples thereof include fibroblasts, hepatocytes, epithelial cells, vascular endothelial cells, mesenchymal stromal cells, mesenchymal stem cells, etc., with dermal fibroblasts, epithelial cells, vascular endothelial cells, mesenchymal stromal cells, and mesenchymal stem cells being preferred, and dermal fibroblasts, vascular endothelial cells, mesenchymal stromal cells, and mesenchymal stem cells being more preferred.

The content of the extracellular vesicles of the present invention in the gene expression regulator of the present invention is not particularly limited as long as the gene expression regulator activity of the present invention (the gene expression regulator effect of the present invention) by the extracellular vesicles of the present invention is exhibited. Furthermore, there are no limitations on the form of the gene expression regulator of the present invention or on the presence or absence of components other than the extracellular vesicles of the present invention.

The gene expression regulator of the present invention contains the extracellular vesicles of the present invention as an active ingredient (main drug), and its dosage form is a solution containing the extracellular vesicles of the present invention as is, or formulated as a liquid, suspension, lipoic agent, etc. together with pharma- ceutically acceptable carriers and additives as necessary, or further formulated as a powder by lyophilization, or as a solid preparation such as a tablet together with pharma-ceutically acceptable additives.

The pharma- ceutically acceptable carriers and additives used in the formulation include the same as those for the cell aging inhibitor of the present invention described above, and the preferred ones are the same.

The gene expression regulator of the present invention is preferably formulated as a liquid, suspension or lipoform, and is basically obtained by mixing a solution containing the extracellular vesicles of the present invention with the above-mentioned carriers and additives as necessary, for example, physiological saline, 5% glucose solution, lipoemulsion, etc. It is also possible to use a lyophilized powder and prepare a preparation that is dissolved or suspended when used.

When the gene expression regulator of the present invention is a liquid, suspension or liposomal agent, its pH is not particularly limited as long as it is within a medicamentously, pharmacologically or physiologically acceptable range, but examples of such ranges include a pH range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0, and can be appropriately adjusted with a pH adjuster.

The administration route of the gene expression regulator of the present invention may be oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, or intraperitoneal administration, depending on the dosage form. The dosage varies depending on the condition of the subject patient (body weight, age, symptoms, physical condition, etc.) and the dosage form, etc., but usually, when administered to an adult, the particle number is 1×10 ⁵ to 1×10 ¹⁷ particles/time, preferably 5×10 ⁵ to 5×10 ¹⁶ particles/time, more preferably 1×10 ⁶ to 1×10 ¹⁶ particles/time, and particularly preferably 5×10 ⁶ to 5×10 ¹⁵ particles/time. This dosage may be administered multiple times a day as a single dosage, or this dosage may be administered in multiple divided doses.

The gene expression regulator of the present invention may be formulated as an external preparation for the skin, such as a transdermal drug or a transdermal quasi-drug. The content of the extracellular vesicles of the present invention in the external preparation for the skin is not particularly limited as long as the gene expression regulator activity of the extracellular vesicles of the present invention is exhibited, and is, for example, preferably 1x10 ⁶ particles/mL to 1x10 ¹⁵ particles/mL, more preferably 1x10 ⁷ particles/mL to 1x10 ¹⁴ particles/mL, and particularly preferably 1x10 ⁸ particles/mL to 1x10 ¹³ particles/mL.

The skin topical preparation may be in any dosage form, for example, a solubilized system such as lotion, a dispersion system such as calamine lotion, or an emulsion system such as cream or milky lotion. It may also be in various dosage forms, such as an aerosol filled with a propellant, a pump spray, an ointment, a cataplasm, a tape, or an injection. In addition to the extracellular vesicles according to the present invention, the skin topical preparation may contain any component (base material) that is usually blended in transdermal pharmaceuticals, transdermal quasi-drugs, etc., depending on the purpose and necessity. Examples of such components include water, oily components, moisturizers, powders, pigments, emulsifiers, solubilizers, gelling agents, detergents, ultraviolet absorbers, anti-inflammatory agents, antiallergic agents, thickeners, pH adjusters, chelating agents, medicines (medicinal components), fragrances, resins, antibacterial and antifungal agents, preservatives (preservatives), antioxidants, slimming agents, alcohols, surfactants, ultraviolet absorbers, moisturizers, emollients, vitamins, colorants, natural extracts, etc. Furthermore, other compounds having the gene expression regulating effect of the present invention can be appropriately blended within the range that does not impair the effect of the present invention. The content of these optional components is not particularly limited, and can be appropriately selected depending on the desired dosage form, application, etc.

The gene expression regulator of the present invention may be produced in accordance with the method for producing the extracellular vesicles of the present invention or the cell aging inhibitor or biological tissue repair promoter of the present invention, and specific examples and preferred methods are also the same.

The present invention will be described in detail below based on examples and comparative examples, but the present invention is not limited to these examples in any way.

Example 1. Obtaining extracellular vesicles from bone marrow-derived mesenchymal stem cells by ultracentrifugation (1) Cell culture Poietics ^TM human mesenchymal stem cells (LONZA), which are bone marrow-derived mesenchymal stem cells, were cultured using MEMα (containing L-glutamine and phenol red, Fujifilm Wako Pure Chemical Industries, Ltd.) containing 15% FBS (Selborne Biological Service Pty., Ltd.) as a growth medium. The cultured bone marrow-derived mesenchymal stem cells were then seeded in a 100 mm cell culture dish (Corning International) at a cell number of 3 x 10 ⁵ , cultured for 72 hours in a cell culture incubator set at 5% CO ₂ and 37°C, and allowed to grow to a cell number of 3 x 10 ⁶ .
(2) Stimulation with Inflammatory Cytokines The bone marrow-derived mesenchymal stem cells proliferated in (1) were stimulated by adding TNFα (R&D Systems) at a final concentration of 20 ng/mL, and further cultured for 24 hours.
(3) Production of extracellular vesicles The bone marrow-derived mesenchymal stem cells cultured in (2) were replaced with 20 mL of D-MEM (FUJIFILM Wako Pure Chemical Industries, Ltd.) containing 10% GIBCO: Fetal Bovine Serum, exosome-depleted, One Shot ^™ format (Thermo Fisher Scientific), which is an extracellular vesicle production medium, and cultured for 120 hours in a cell culture incubator set at 5% CO ₂ and 37° C. Thereafter, the obtained culture supernatant was collected in a 50 mL centrifuge tube and centrifuged at 2000×g for 20 minutes, and the supernatant was collected.
(4) Obtaining extracellular vesicles by ultracentrifugation Next, the culture supernatant collected in (3) was centrifuged at 110,000×g for 70 minutes using an ultracentrifuge (Beckman: Optima ^™ L-100XP). 1 mL of PBS was added to the resulting pellet, and the mixture was centrifuged again at 110,000×g for 70 minutes. The final pellet was dissolved in PBS containing EV-Save ^™ extracellular vesicle blocking reagent (FUJIFILM Wako Pure Chemical Industries, Ltd.). Hereinafter, the resulting solution may be referred to as "extracellular vesicle solution (bone marrow-derived MSC, ultracentrifugation, TNFα stimulation)".

Example 2. Obtaining extracellular vesicles from umbilical cord-derived mesenchymal stem cells by ultracentrifugation. Extracellular vesicles were obtained by ultracentrifugation in the same manner as in Example 1, except that "Human ^Mesenchymal Stem Cells from Umbilical Cord Matrix (PromoCell)", which are mesenchymal stem cells derived from the umbilical cord, was used instead of "Poietics TM human mesenchymal stem cells (LONZA)". Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (umbilical cord-derived MSC, ultracentrifugation, TNFα stimulation)".

Comparative Example 1. Obtaining extracellular vesicles from bone marrow-derived mesenchymal stem cells by ultracentrifugation Extracellular vesicles were obtained by ultracentrifugation in the same manner as in Example 1, except that "Example 1 (2) Stimulation with inflammatory cytokines" was not performed. Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (bone marrow-derived MSC, ultracentrifugation)".

Comparative Example 2. Obtaining extracellular vesicles from umbilical cord-derived mesenchymal stem cells by ultracentrifugation. Extracellular vesicles were obtained by ultracentrifugation in the same manner as in Comparative Example 1, except that "Human ^Mesenchymal Stem Cells from Umbilical Cord Matrix (PromoCell)" which is an umbilical cord-derived mesenchymal stem cell was used instead of "Poietics TM human mesenchymal stem cells (LONZA)". Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (umbilical cord-derived MSC, ultracentrifugation)".

Example 3. Acquisition of extracellular vesicles from bone marrow-derived mesenchymal stem cells by PS affinity method Extracellular vesicles were acquired by the same method as in Example 1, except that extracellular vesicles were acquired by the PS affinity method according to the following procedure instead of "Example 1 (4) Acquisition of extracellular vesicles by ultracentrifugation method". Extracellular vesicles were isolated from 1 mL of the culture supernatant collected in Example 1 (3) using MagCapture ^TM Exosome Isolation Kit PS (FUJIFILM Wako Pure Chemical Industries, Ltd.) according to the procedure described in the instructions attached to the kit, and the extracellular vesicles were obtained in 1 mM EDTA-containing PBS (phosphate-buffered saline) to which EV-Save ^TM extracellular vesicle blocking reagent (FUJIFILM Wako Pure Chemical Industries, Ltd.) was added. Thereafter, the buffer was exchanged with PBS containing EV-Save ^™ extracellular vesicle blocking reagent using Vivaspin 500 (Sartorius, molecular weight cutoff: 100,000 (100K), membrane material: PES). Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (bone marrow-derived MSC, PS method, TNFα stimulation)".

Example 4. Acquisition of extracellular vesicles from umbilical cord-derived mesenchymal stem cells by the PS affinity method. Extracellular vesicles were obtained by the PS method in the same manner as in Example 3, except that "Human ^Mesenchymal Stem Cells from Umbilical Cord Matrix (PromoCell)", which are umbilical cord-derived mesenchymal stem cells, were used instead of "Poietics TM human mesenchymal stem cells (LONZA)". Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (umbilical cord-derived MSC, PS method, TNFα stimulation)".

Example 5. Acquisition of extracellular vesicles from umbilical cord-derived mesenchymal stem cells by PS affinity method "Umbilical cord-derived mesenchymal stem cells, Human Mesenchymal Stem Cells from Umbilical Cord Matrix (PromoCell)" was used instead of "Poietics ^TM human mesenchymal stem cells (LONZA)" and extracellular vesicles were obtained by the PS method in the same manner as in Example 1, except that "Example 1 (2) Stimulation with inflammatory cytokines" was not performed. Hereinafter, the obtained solution may be referred to as "extracellular vesicle solution (umbilical cord-derived MSC, PS method)".

Experimental Example 1-7. Measurement of extracellular vesicle particle number by nano tracking analysis method The particle number per unit volume of the "extracellular vesicle solution" obtained in each of Examples 1-5 and Comparative Example 1-2 was measured by nanoparticle tracking analysis (Nano Tracking Analysis) using NanoSight (Malvern Panalytical Co.) according to the procedure described in the NanoSight instruction manual, and the average particle size and the average particle number per unit volume [particles / mL] were calculated. The "extracellular vesicle solution" used in the measurement and the obtained average particle size and average particle number per unit volume are shown in Table 1 below. In addition, a graph of the obtained particle size distribution is shown in Figure 1 together with the results of Experimental Example 2. In the graph of Figure 1, the vertical axis indicates the number of particles and the horizontal axis indicates the particle size.

Example 6-11. Evaluation of the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles obtained by the PS affinity method Using the "extracellular vesicle solution (bone marrow-derived MSCs, ultracentrifugation, TNFα stimulation)" obtained in Example 1 and the "extracellular vesicle solution (bone marrow-derived MSCs, PS method, TNFα stimulation)" obtained in Example 3, the mRNA expression levels of the p21 gene, p53 gene, and MMP-1 gene, which are cellular senescence-related genes and biological tissue repair-related genes, were measured by quantitative PCR, to evaluate the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of the extracellular vesicles. Specifically, this was carried out by the following method. TIG3 cells (distributed by JCRB, after division 70 times or more), which are human fetal lung-derived fibroblasts, were suspended in DMEM (FUJIFILM Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Biosera Co., Ltd.), and seeded in a 96-well plate (Corning Co., Ltd.) at 2 x 10 ³ cells per well and 100 μL of medium. 16 hours after cell seeding, the "extracellular vesicle solution" described in Table 2 below was added to each well in an amount to a final concentration of 3 x 10 ⁹ particles/mL, and cultured for 96 hours. RNA was extracted using the PureLink ^TM RNA Mini Kit (ThermoFisher Scientific Co., Ltd.) according to the procedure described in the instructions attached to the kit. From 50 ng of extracted RNA, cDNA was synthesized using ReverTra Ace ^™ qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.) according to the procedure described in the instructions attached to the kit. Using the synthesized cDNA, the mRNA expression levels of p21 gene, p53 gene, MMP-1 gene, and GAPDH, which are cell aging-related genes and biological tissue repair-related genes, and the internal standard, were measured by quantitative PCR using KOD SYBR qPCR Mix (Toyobo Co., Ltd.). The primers used for quantitative PCR are shown in FIG. 3.
Figure 4 shows the results for the mRNA expression level of the p21 gene, Figure 5 shows the results for the mRNA expression level of the p53 gene, and Figure 6 shows the results for the mRNA expression level of the MMP-1 gene. The expression level of each gene was normalized to GAPDH from the values obtained by qPCR performed using primers for each gene, and shown as a relative value (△△ct value) to the control, which was used as the vertical axis of each figure. In the figure, the horizontal axis "EV (-) purification method (-)" indicates the results when TIG3 cells (control) without EV were used, "EV (MSC) purification method (UC)" indicates the results when "extracellular vesicle solution (bone marrow-derived MSC, ultracentrifugation)" obtained in Comparative Example 1 was used, "EV (MSC TNFα stimulation) purification method (UC)" indicates the results when "extracellular vesicle solution (bone marrow-derived MSC, ultracentrifugation, TNFα stimulation)" obtained in Example 1 was used, and "EV (MSC) purification method (PS)" indicates the results when "extracellular vesicle solution (bone marrow-derived MSC, PS method, TNFα stimulation)" obtained in Example 3 was used. The extracellular vesicle solution used for measurement in each example, the mRNA whose expression level was confirmed, and the number of the figure showing the results are shown in Table 2 below, together with those of Comparative Examples 3-5.

Comparative Example 3-5. Evaluation of the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles obtained by ultracentrifugation The cellular senescence-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles were evaluated by measuring the mRNA expression levels of p21 gene, p53 gene, and MMP-1 gene, which are cellular senescence-related genes and biological tissue repair-related genes, by quantitative PCR in the same manner as in Examples 6-11, except that the "extracellular vesicle solution (bone marrow-derived MSC, ultracentrifugation)" obtained in Comparative Example 1 was used as the "extracellular vesicle solution". The extracellular vesicle solutions used for the measurement in each comparative example and the mRNAs whose expression levels were confirmed are as shown in Table 2 above. The results of quantitative PCR are shown in Figures 4-6, respectively, together with the results of Examples 6-11.

Examples 12-18. Evaluation of the cellular senescence inhibitory activity and biological tissue repair promoting activity of extracellular vesicles obtained by the PS affinity method As the "extracellular vesicle solution", the "extracellular vesicle solution (umbilical cord-derived MSC, ultracentrifugation, TNFα stimulation)" obtained in Example 2, the "extracellular vesicle solution (umbilical cord-derived MSC, PS method, TNFα stimulation)" obtained in Example 4, and the "extracellular vesicle solution (umbilical cord-derived MSC, PS method)" obtained in Example 5 were used in an amount to give a final concentration of 7.5 × 10 ⁹ particles / mL, except that the mRNA expression levels of the p21 gene, p53 gene, and MMP-1 gene, which are cellular senescence-related genes and biological tissue repair-related genes, were measured by quantitative PCR in the same manner as in Example 6-11 to evaluate the cellular senescence inhibitory activity and biological tissue repair promoting activity of the extracellular vesicles. The extracellular vesicle solution used for the measurement in each example, the mRNA whose expression level was confirmed, and the number of the figure showing the results are shown in Table 3 below, along with the conditions of Comparative Example 6-8. The results of quantitative PCR are shown in Figures 7 to 9, together with the results of Comparative Examples 6 to 8, respectively.

Comparative Example 6-8. Evaluation of the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of extracellular vesicles obtained by ultracentrifugation The "extracellular vesicle solution (umbilical cord-derived MSC, ultracentrifugation)" obtained in Comparative Example 2 was used as the "extracellular vesicle solution" in an amount to a final concentration of 7.5 x 10 ⁹ particles/mL. The mRNA expression levels of the p21 gene, p53 gene, and MMP-1 gene, which are cellular senescence-related genes and biological tissue repair-related genes, were evaluated by measuring them by quantitative PCR in the same manner as in Example 6-11. The extracellular vesicle solutions used for the measurement in each comparative example and the mRNAs whose expression levels were confirmed are as shown in Table 3 above. The results of quantitative PCR are shown in Figures 7-9, respectively, along with the results of Examples 12-18.

Figures 4-9 show that extracellular vesicles obtained from mesenchymal stem cells stimulated with inflammatory cytokines and extracellular vesicles obtained from mesenchymal stem cells by the PS affinity method suppressed the mRNA expression levels of cellular senescence-related genes and biological tissue repair-related genes more than extracellular vesicles obtained from mesenchymal stem cells by ultracentrifugation, regardless of the type of mesenchymal stem cell. In other words, it was found that the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of the obtained extracellular vesicles are enhanced by stimulating mesenchymal stem cells with inflammatory cytokines, and that the extracellular vesicles obtained from mesenchymal stem cells by the PS method have higher cellular senescence-inhibiting activity and biological tissue repair-promoting activity than the extracellular vesicles obtained from mesenchymal stem cells by ultracentrifugation. In particular, it was found that the extracellular vesicles obtained by the PS method from mesenchymal stem cells stimulated with inflammatory cytokines have significantly higher cellular senescence-inhibiting activity and biological tissue repair-promoting activity.

Examples 19-28 and Comparative Example 9. Acquisition of extracellular vesicles from mesenchymal stem cells Extracellular vesicles were acquired by the same methods as in Examples 1-5 and Comparative Examples 1-2, except that the conditions (mesenchymal stem cells, acquisition method, stimulation) described in Table 4 below were followed. In Table 4, "bone marrow-derived MSC" refers to "Poietics ^TM human mesenchymal stem cells (LONZA Corporation) which are bone marrow-derived mesenchymal stem cells,""umbilical cord-derived MSC" refers to "Human Mesenchymal Stem Cells from Umbilical Cord Matrix (PromoCell Corporation) which are umbilical cord-derived mesenchymal stem cells,""adipose-derivedMSC" refers to "Human Adipose-Derived Stem Cells (LONZA Corporation) which are adipose-derived mesenchymal stem cells,""PSmethod" refers to the PS affinity method, and "stimulation" refers to stimulation by inflammatory cytokines.

Examples 29-69 and Comparative Examples 10-26. Evaluation of Cellular Senescence Inhibitory Activity and Biological Tissue Repair Promoting Activity of Extracellular Vesicles Using the extracellular vesicle solutions obtained in Examples 19-28 and Comparative Example 9, the mRNA expression levels of the cellular senescence-related genes and biological tissue repair-related genes, p16 gene, p21 gene, MMP-1 gene, type I collagen gene (COL1A1 gene), type III collagen gene (COL3A1 gene), and HAS1 gene, were measured by quantitative PCR to evaluate the cellular senescence-inhibitory activity and biological tissue repair promoting activity of the extracellular vesicles. Specifically, the following method was used. TIG3S cells (distributed by JCRB, after division 50 times or more), which are human skin-derived fibroblasts, were suspended in DMEM (FUJIFILM Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Biosera Co., Ltd.), and seeded in a 96-well plate (Corning Co., Ltd.) at 3 x 10 ³ cells per well and 100 μL of medium. 16 hours after cell seeding, the "extracellular vesicle solution" described in Tables 5 and 6 below was added to each well in an amount to a final concentration of 2 x 10 ⁸ particles/mL, and cultured for 96 hours. Then, RNA was extracted using a PureLink ^TM RNA Mini kit (ThermoFisher Scientific Co., Ltd.) according to the procedure described in the instructions attached to the kit. From 50 ng of the extracted RNA, cDNA was synthesized using ReverTra Ace ^™ qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.) according to the procedure described in the instructions attached to the kit. Using the synthesized cDNA, the mRNA expression levels of the p16 gene, p21 gene, MMP-1 gene, type I collagen gene (COL1A1 gene), type III collagen gene (COL3A1 gene), HAS1 gene, and internal standard GAPDH were measured by quantitative PCR using KOD SYBR qPCR Mix (Toyobo Co., Ltd.). The sequences of the primers used are shown in FIG. 10. Fig. 11 shows the mRNA expression level of p16 gene, Fig. 12 shows the mRNA expression level of p21 gene, Fig. 13 shows the mRNA expression level of MMP-1 gene, Fig. 14 shows the mRNA expression level of type I collagen gene, Fig. 15 shows the mRNA expression level of type III collagen gene, and Fig. 16 shows the mRNA expression level of HAS1 gene. The expression level of each gene was normalized to GAPDH from the values obtained by qPCR performed using primers for each gene, and shown as a relative value (△△ct value) to the control, which was used as the vertical axis of each figure. In the figure, the horizontal axis indicates "Purification method (-) Extracellular vesicles (-)" for the results when TIG3S cells (control) without the addition of extracellular vesicles were used, "UC" in "Purification method" indicates ultracentrifugation, "PS" indicates the PS affinity method, and "MSC" in "Extracellular vesicles" indicates no stimulation with inflammatory cytokines, "MSC TNFα" indicates stimulation with TNFα, and "MSC IL-1β" indicates stimulation with IL-1β. The mRNAs whose expression levels were confirmed in each Example and Comparative Example, and the numbers of the figures showing the results, are shown in Tables 5 and 6 below, respectively.

Figures 11-14 show that extracellular vesicles obtained from mesenchymal stem cells stimulated with inflammatory cytokines and extracellular vesicles obtained from mesenchymal stem cells by the PS affinity method suppressed the mRNA expression levels of cellular senescence-related genes and biological tissue repair-related genes more than extracellular vesicles obtained from mesenchymal stem cells by ultracentrifugation, regardless of the type of mesenchymal stem cell. In other words, it was found that the cellular senescence-inhibiting activity and biological tissue repair-promoting activity of the obtained extracellular vesicles are enhanced by stimulating mesenchymal stem cells with inflammatory cytokines, and that the extracellular vesicles obtained from mesenchymal stem cells by the PS method have higher cellular senescence-inhibiting activity and biological tissue repair-promoting activity than the extracellular vesicles obtained from mesenchymal stem cells by ultracentrifugation. In particular, it was found that the extracellular vesicles obtained by the PS method from mesenchymal stem cells stimulated with inflammatory cytokines have significantly higher cellular senescence-inhibiting activity and biological tissue repair-promoting activity.

The present invention provides a cell aging inhibitor, a biological tissue repair promoter, a gene expression regulator, and a skin topical composition, which contain as active ingredients extracellular vesicles having a cell aging inhibitory effect from mesenchymal stem cells. The cell aging inhibitor and skin topical composition provided by the present invention regulate the gene expression of cell aging-related genes and biological tissue repair-related genes, and have great industrial applicability in that cell aging is inhibited by cell proliferation promoting action, collagen production promoting or decomposition inhibiting action, hyaluronic acid production promoting or decomposition inhibiting action, elastin production promoting or decomposition inhibiting action, etc. Furthermore, the gene expression regulator provided by the present invention regulates the expression of at least one gene selected from a cell proliferation inhibitory gene, a collagen gene, a collagen decomposition enzyme gene, a hyaluronic acid synthase gene, a hyaluronic acid decomposition enzyme gene, an elastin gene, and an elastin decomposition enzyme gene. The biological tissue repair promoter provided by the present invention can, for example, promote the function of cells, biological tissues, and organs that have become dysfunctional or have malfunctioned by promoting cell proliferation, promoting the production of collagen or inhibiting its degradation, promoting the production of hyaluronic acid or inhibiting its degradation, and promoting the production of elastin or inhibiting its degradation.

Claims (10)

A cellular aging inhibitor or a biological tissue repair promoter, comprising extracellular vesicles derived from mesenchymal stem cells as active ingredients,
the extracellular vesicles are derived from mesenchymal stem cells stimulated with an inflammatory cytokine, phosphatidylserine is exposed on the membrane surface of the extracellular vesicles, and Tim protein is capable of specifically binding to the phosphatidylserine;
the inflammatory cytokine is tumor necrosis factor alpha or interleukin 1;
A cellular aging inhibitor or a biological tissue repair promoter, in which the inhibition of cellular aging or the promotion of biological tissue repair is achieved by at least one action selected from the action of promoting cell proliferation, the action of promoting collagen production or inhibiting collagen decomposition, and the action of promoting hyaluronic acid production . The cell aging inhibitor or biological tissue repair promoter according to claim 1, wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane, and placenta. The cell aging inhibitor or biological tissue repair promoter according to claim 1, wherein the Tim protein is selected from Tim4 protein, Tim3 protein, and Tim1 protein. The method includes obtaining extracellular vesicles from mesenchymal stem cells stimulated with inflammatory cytokines, and obtaining extracellular vesicles from the extracellular vesicles by a method using a substance having affinity for phosphatidylserine,
the inflammatory cytokine is tumor necrosis factor alpha or interleukin 1;
The inhibition of cellular aging or the promotion of biological tissue repair is due to at least one action selected from the group consisting of the action of promoting cell proliferation, the action of promoting collagen production or inhibiting collagen decomposition, and the action of promoting hyaluronic acid production.
A method for producing extracellular vesicles having the effect of inhibiting cellular aging or promoting the repair of biological tissue. The method for producing extracellular vesicles having an effect of inhibiting cellular aging or promoting the repair of biological tissue according to claim 4 , wherein the mesenchymal stem cells are derived from iPS cells or from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, dental pulp, amniotic membrane and placenta. The method for producing extracellular vesicles having an effect of inhibiting cellular aging or promoting the repair of biological tissue, as described in claim 4 , wherein the substance having affinity for phosphatidylserine is a Tim protein. The method for producing extracellular vesicles having an effect of inhibiting cellular aging or promoting the repair of biological tissue, as described in claim 6 , wherein the Tim protein is selected from the group consisting of Tim4 protein, Tim3 protein and Tim1 protein. A method for producing a cellular aging inhibitor or a biological tissue repair promoter, comprising incorporating, as an active ingredient, extracellular vesicles obtained by the method for producing extracellular vesicles according to any one of claims 4 to 7. A gene expression regulator comprising extracellular vesicles derived from mesenchymal stem cells as active ingredients,
the extracellular vesicles are derived from mesenchymal stem cells stimulated with an inflammatory cytokine, and phosphatidylserine is exposed on the membrane surface of the extracellular vesicles, and the Tim protein is capable of specifically binding to the phosphatidylserine; and the gene is at least one gene selected from a cell proliferation inhibitory gene, a collagen gene, a collagen decomposition enzyme gene, and a hyaluronic acid synthase gene,
The gene expression regulator , wherein the inflammatory cytokine is tumor necrosis factor α or interleukin-1 . A composition for external application to the skin for inhibiting cell aging or promoting biological tissue repair, comprising the cell aging inhibitor or biological tissue repair promoter according to any one of claims 1 to 3 .

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