Cell preparation used for suppressing muscle mass loss

Abstract

To provide a cell preparation that can suppress muscle mass loss.SOLUTION: The present invention pertains to a cell preparation used for suppressing muscle mass loss, the cell preparation containing umbilical cord-derived cells.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This invention relates to a cell preparation used to suppress the decrease in muscle mass. [Background technology]

[0002] In Japan, the population is aging rapidly, and it is projected that by 2025, the number of elderly people will exceed 30% of the total population. As people age, various functional impairments occur. One of the causes of these functional impairments is age-related muscle loss (age-related muscle loss). Sarcopenia is one type of age-related muscle loss. The presence of sarcopenia is closely related to "dizziness," "falls," and "frailty" in the elderly, and it is known that this can lead to a state of needing long-term care, which is a cause for concern (Non-patent Literature 1). To date, improving nutritional status, increasing daily physical activity, and engaging in light exercise have been recommended, but these are not easy for many elderly people to implement, and new therapeutic interventions are needed. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Masafumi Kuzuya, "3. Diagnosis, Pathophysiology, and Treatment of Sarcopenia," 2015, Journal of the Japan Geriatrics Society, Vol. 52, No. 4, pp. 343-349. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Treatment options for age-related muscle loss include exercise and nutritional interventions. However, it is currently unclear whether either method is sufficiently effective.

[0005] Therefore, the present invention aims to provide a cell preparation that can suppress the decrease in muscle mass. [Means for solving the problem]

[0006] To achieve the above object, the present invention provides a cell preparation for suppressing muscle mass reduction, wherein the cell preparation contains cord-derived cells.

[0007] The present invention provides a cell preparation for treating sarcopenia, which contains the cell preparation for suppressing muscle mass reduction according to the present invention.

Advantages of the Invention

[0008] According to the present invention, muscle mass reduction can be suppressed.

Brief Description of the Drawings

[0009] [Figure 1] Figure 1 is a graph showing the change in body weight in Example 1. [Figure 2] Figure 2 is a graph showing the change in grip strength per body weight in Example 1. [Figure 3] Figure 3 is a graph showing the endurance in Example 1. [Figure 4] Figure 4 is a graph showing the measurement result of muscle weight in Example 1. [Figure 5] Figure 5 is a photograph showing the staining result of muscle tissue in Example 2. [Figure 6] Figure 6 is a graph showing the expression levels of PGC1-α, COX4, and GLUT4 in the gastrocnemius muscle and soleus muscle of each mouse in Example 3. [Figure 7] Figure 7 is a photograph showing the result of Western blot in Example 3. [Figure 8] Figure 8 is a photograph and a graph showing the measurement result of mitochondria in Example 3. [Figure 9] Figure 9 is a photograph and a graph showing the result of apoptosis analysis in Example 4. [Figure 10]Figure 10 shows a graph illustrating the expression levels of inflammatory cytokines in Example 5, as well as a photograph of muscle tissue. [Figure 11] Figure 11 shows a graph and a photograph of muscle tissue illustrating the expression level of TGF-β1 in Example 6. [Figure 12] Figure 12 is a graph or photograph showing the expression levels of CatK, Sirt1, and MHC in Example 7. [Figure 13] Figure 13 is an electron microscope image of exosomes in Example 8. [Figure 14] Figure 14 is a photograph of the exosome marker obtained by Western blotting in Example 8. [Figure 15] Figure 15 is a graph showing the particle size distribution of the exosome marker in Example 8. [Figure 16] Figure 16 is a photograph showing C2C12 cells that have taken up exosomes in Example 8. [Figure 17] Figure 17 shows a photograph and graph illustrating apoptosis in C2C12 cells that incorporated exosomes in Example 8. [Modes for carrying out the invention]

[0010] <Preparation for suppressing muscle mass loss> As described above, the present invention relates to a cell preparation used to suppress the decrease in muscle mass (hereinafter also referred to as the "preparation for suppressing the decrease in muscle mass"), wherein the cell preparation includes umbilical cord-derived cells. The preparation for suppressing the decrease in muscle mass of the present invention is characterized by including umbilical cord-derived cells, and other components and conditions are not particularly limited. The umbilical cord-derived cells are presumed to have functions such as improving the functionality of mitochondria in muscles, muscle tissue, or muscle cells (also referred to as "muscle fibers" or "muscle cells," the same applies hereinafter), suppressing inflammation, suppressing apoptosis, promoting the proliferation of muscle cells, suppressing muscle damage, suppressing interstitial fibrosis of muscle tissue, and / or promoting muscle repair. For this reason, the preparation for suppressing the decrease in muscle mass of the present invention can suppress the decrease in muscle mass by including the umbilical cord-derived cells.

[0011] In the present invention, "muscle mass" refers to, for example, the amount of muscle in the limbs of a subject, and a specific example is the amount of gastrocnemius muscle. In the present invention, the "muscle mass" may be evaluated, for example, by measuring the weight of the muscle of the subject to be measured, or by indirectly calculating the volume using images obtained using dual-energy X-ray absorptiometry (DEXA), bioelectrical impedance analysis, computed tomography (CT), or magnetic resonance imaging (MRI). When the subject to be measured is a mammal other than a human, it is preferable to evaluate the muscle mass by measuring the weight of the muscle of the subject. On the other hand, when the subject to be measured is a human, it is preferable to evaluate the muscle mass by indirectly calculating the volume using dual-energy X-ray absorptiometry (DEXA), bioelectrical impedance analysis, etc.

[0012] In the present invention, "suppression of muscle mass decline" means that the decline in muscle mass (also referred to as "decrease," "regression," or "reduction," hereinafter the same) is significantly suppressed (also referred to as "inhibition," "blockage," or "prevention," hereinafter the same). Specifically, "suppression of muscle mass decline" means, for example, that the degree of muscle mass decline is significantly suppressed in subjects who are administered the cell preparation compared to subjects who are not administered the cell preparation. Therefore, in the present invention, even if the muscle mass of subjects who are administered the cell preparation has decreased compared to the time of administration of the cell preparation, if the degree of muscle mass decline is significantly suppressed compared to subjects who are not administered the cell preparation, it can be said that "suppression of muscle mass decline" has occurred.

[0013] The cell preparations of the present invention are presumed to exhibit an effect of suppressing the decrease in muscle mass by increasing muscle mass, as shown in the examples described below. For this reason, the cell preparations used to suppress the decrease in muscle mass of the present invention can also be called, for example, cell preparations used to increase muscle mass (also referred to as "increase," "enhance," or "increase in volume," hereinafter the same).

[0014] As shown in the examples described below, the cell preparations of the present invention suppress the decline in muscle strength, such as sustained muscle strength, or promote the increase of such muscle strength. This is presumed to be due to the cell preparations of the present invention exhibiting functions such as suppressing the decline in muscle mass. For this reason, the cell preparations used for suppressing the decline in muscle mass in the present invention can also be described as cell preparations used for suppressing or increasing (also referred to as "increasing" or "enhancing" muscle strength; the same applies hereinafter) muscle strength.

[0015] In this invention, "umbilical cord" refers to a white, tubular tissue that connects the fetus to the placenta, and does not include the placenta or umbilical cord blood. In this invention, the origin of the "umbilical cord" is not particularly limited, and examples include the umbilical cords of mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. Preferably, it is the umbilical cord of a primate mammal, and more preferably, it is the umbilical cord of a human.

[0016] In the present invention, the "umbilical cord" may be an umbilical cord collected from a subject to administration, treatment, or procedure (hereinafter collectively referred to as the "administered subject"), or an umbilical cord collected from a subject other than the administered subject. From the viewpoint of not being restricted during preparation, it is desirable to use an umbilical cord collected from a subject other than the administered subject. The umbilical cord-derived cells of the present invention may be cells derived from an umbilical cord collected from a subject other than the administered subject, as shown in the examples described later. It has been confirmed that the umbilical cord-derived cells exert a therapeutic effect without being rejected, for example, by immune rejection.

[0017] In the present invention, the umbilical cord can be recovered, for example, from the placenta and / or umbilical cord delivered by vaginal delivery or cesarean section, by appropriately removing the placenta from the postpartum tissue. In the present invention, the umbilical cord may be one from which umbilical cord blood has been removed, and may also be sterilized or bacteriostatically treated. The removal of umbilical cord blood can be carried out, for example, by rinsing or perfusing with a solution containing an anticoagulant such as heparin. The sterilization or bacteriostatic treatment is not particularly limited and can be carried out, for example, by applying a disinfectant such as povidone-iodine; immersion in a culture medium or buffer to which antibiotics such as penicillin, streptomycin, amphotericin B, gentamicin, and / or nystatin, and / or antifungal agents have been added; and so on. In addition, the umbilical cord may be selectively lysed with red blood cells, for example, if necessary. As a method for selectively lysing the aforementioned red blood cells, well-known methods in the art, such as incubation in a hypertonic or hypotonic medium by dissolution with ammonium chloride, can be used.

[0018] In the present invention, the term "umbilical cord-derived cells" refers to a population of cells prepared using umbilical cord as a raw material.

[0019] The umbilical cord-derived cells of the present invention may be, for example, a cell population having one or more of the following characteristics (a) to (c), and preferably a cell population having all of the characteristics. (a) When cultured in the presence of culture medium, it exhibits adhesion to plastic; (b) Positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L (Programmed cell death 1 ligand) 2, and negative for CD45, CD34, CD11b, CD19, and HLA-Class II; (c) Under inflammatory conditions, the expression of the IDO (indoleamine 2,3-dioxygenase), PGE2 (Prostaglandin E2), and PD-L1 genes and / or proteins is induced.

[0020] In the present invention, "positive" means that a high signal, etc., is detected by an analytical method such as flow cytometry that utilizes the antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. Also, in the present invention, "negative" means that a signal, etc., equivalent to or lower than that detected by a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0021] In this invention, "HLA-class I" means HLA-A, B, or C. In this invention, "HLA-Class II" means HLA-DR, DQ, or DP.

[0022] In the present invention, "under inflammatory conditions" refers to conditions in which an inflammatory cytokine such as interferon-γ is brought into contact with or added to the substance.

[0023] In the present invention, the umbilical cord-derived cells may be extracts and / or secretions of the umbilical cord-derived cells. Examples of extracts of umbilical cord-derived cells include products obtained by concentration, centrifugation, drying, freeze-drying, solvent treatment, surfactant treatment, enzymatic treatment using proteases, glycosylases, etc., protein extraction, sonication, and / or grinding of the umbilical cord-derived cells, or products obtained by a combination of these treatments. Examples of secretions of umbilical cord-derived cells include exosomes (extracellular vesicles) and the supernatant of cell culture of umbilical cord-derived cells.

[0024] In this invention, "extracellular vesicle" refers to a membrane-containing vesicle secreted from a cell. These extracellular vesicles are generally thought to be formed within the endosomes of the originating cell and then released outside the cell. Therefore, these extracellular vesicles typically include a lipid bilayer and a lumen, the lumen having a structure surrounded by the lipid bilayer. The lipid bilayer contains lipids derived from the cell membrane of the originating cell. The lumen contains cytoplasm derived from the originating cell. These extracellular vesicles are classified according to their size and / or surface markers into, for example, exosomes, microvesicles (MVs), apoptotic bodies, etc.

[0025] The average diameter (weighted average) of the extracellular vesicles is, for example, 1 to 500 nm, preferably 10 to 400 nm, and more preferably 30 to 400 nm. The average diameter can be measured according to Example 8 described later. The average diameter of the extracellular vesicles can also be adjusted, for example, by filtering the liquid containing the extracellular vesicles using a filter with a desired pore size.

[0026] In the present invention, it is preferable that the cell preparation exhibits an inhibitory effect on interstitial fibrosis in muscle tissue. The inhibitory effect on interstitial fibrosis can be evaluated, for example, using the fibrotic area in the interstitium of muscle tissue as an indicator, according to Example 2 described below. Specifically, it can be evaluated using the fibrotic area in the interstitium of muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice as an indicator (interstitial fibrosis assay). In the above evaluation, for example, if the interstitial fibrosis area in the group administered the test substance is 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less compared to the interstitial fibrosis area in the group not administered the test substance, then the test substance can be evaluated as having an inhibitory effect on interstitial fibrosis in muscle tissue. The cell preparation can reduce interstitial fibrosis by 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less, compared to the group administered the cell preparation in the interstitial fibrosis assay. The preparation for suppressing muscle mass reduction of the present invention can suppress interstitial fibrosis in muscle tissue, and therefore can also be described as a cell preparation used to suppress interstitial fibrosis in muscle tissue, for example.

[0027] In this invention, "muscle tissue" refers to tissue composed of muscle cells (muscle fibers), and is, for example, an excitable tissue that has contractile ability.

[0028] In this invention, "interstitium" refers to the space between muscle fibers.

[0029] In the present invention, it is preferable that the cell preparation exhibits a function-enhancing effect on mitochondria. This function-enhancing effect on mitochondria can be evaluated, for example, according to Example 3 described below, using the expression levels of mitochondrial function-enhancing genes such as PGC1-α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), COX4 (cytochrome c oxidase subunit 4), and GLUT4 (glucose transporter type 4) in muscle tissue, or the number of mitochondria in muscle tissue, as indicators. Specifically, it can be evaluated using the expression levels of mitochondrial function-enhancing genes in mitochondria in the muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice, or the number of mitochondria in muscle tissue (e.g., gastrocnemius or soleus muscle) as indicators (mitochondrial function assay). In the above evaluation, for example, if the expression level of mitochondrial function-enhancing genes in the group administered the test substance is 1.5 times or more, 2 times or more, 2.5 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, 100 times or more, 125 times or more, or 250 times or more compared to the expression level of mitochondrial function-enhancing genes in the group not administered the test substance, then the test substance can be evaluated as having a mitochondrial function-enhancing effect. Also, in the above evaluation, for example, if the number of mitochondria in the group administered the test substance is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 100% or more compared to the number of mitochondria in the group not administered the test substance, then the test substance can be evaluated as having a mitochondrial function-enhancing effect. The cell preparation can, for example, in the mitochondrial function assay, increase the expression level of the mitochondrial function-enhancing gene by 1.5 times or more, 2 times or more, 2.5 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, 100 times or more, 125 times or more, or 250 times or more compared to the group administered the cell preparation, with an upper limit of, for example, 1000 times or less or 500 times or less.The cell preparation can increase the number of mitochondria by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 100% or more compared to the group administered the cell preparation, with the upper limit being, for example, 150% or less or 125% or less. The preparation for suppressing muscle mass decline of the present invention improves mitochondrial function in muscle tissue, and therefore can also be described as, for example, a cell preparation used to improve mitochondrial function in muscle tissue. Furthermore, since the preparation for suppressing muscle mass decline of the present invention can induce the expression of genes related to mitochondrial functionality in muscle tissue, it can also be described as, for example, a cell preparation used to induce (induce expression of) genes that improve mitochondrial function in muscle tissue. Furthermore, since the preparation for suppressing muscle mass loss according to the present invention can increase the number of mitochondria in muscle tissue, it can also be described as a cell preparation used to increase the number of mitochondria in muscle tissue, for example.

[0030] The mRNA sequences and protein amino acid sequences of human PGC1-α, human COX4, and human GLUT4 can be referenced, for example, from sequences registered under the following Genbank accession numbers. Human PGC1-α mRNA accession number: NM_001330751 Protein accession number: NP_001317680 • Human COX4 mRNA accession number: NM_001861 Protein accession number: NP_001852 • Human GLUT4 mRNA accession number: NM_001042 Protein accession number: NP_001033

[0031] In the present invention, it is preferable that the cell preparation exhibits an inhibitory effect on myocyte apoptosis. The inhibitory effect on myocyte apoptosis can be evaluated, for example, according to Example 4 described below, using the expression levels of cleaved-caspase-3 and / or cleaved-caspase-8, or DNA fragmentation (genomic DNA fragmentation) as indicators. Specifically, it can be evaluated using the number of apoptotic cells in the muscle tissue of SAMP10 mice (e.g., gastrocnemius or soleus muscle) as an indicator (apoptosis assay). In the above evaluation, for example, if the number of apoptotic cells in the group administered the test substance is 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less compared to the number of apoptotic cells in the group not administered the test substance, then the test substance can be evaluated as having an inhibitory effect on muscle cell apoptosis. The cell preparation can reduce the number of apoptotic cells by 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less in the apoptotic assay described above. The preparation for suppressing muscle mass reduction of the present invention exhibits suppression of apoptosis in muscle cells, and therefore can also be described as a cell preparation used to suppress apoptosis in muscle cells, for example. The muscle cells are, for example, skeletal muscle cells.

[0032] In the present invention, it is preferable that the cell preparation exhibits anti-inflammatory activity. This anti-inflammatory activity can be evaluated, for example, by the ability to induce inflammatory cytokine genes or chemokine genes, as described in Example 5 below. Specifically, it can be evaluated by the expression level of inflammatory cytokine genes or chemokine genes in the muscle tissue of SAMP10 mice (e.g., gastrocnemius or soleus muscle) (inflammation assay). In the above evaluation, for example, if the expression level of inflammatory cytokine genes or chemokine genes in the group administered the test substance is 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less compared to the expression level of inflammatory cytokine genes or chemokine genes in the group not administered the test substance, then the test substance can be evaluated as having an anti-inflammatory effect. The cell preparation can reduce the expression levels of inflammatory cytokine genes and / or chemokine genes to 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, 97% or less, 98% or less, or 99% or less in the inflammation assay compared to the group administered the cell preparation. Since the preparation for suppressing muscle mass reduction of the present invention exhibits anti-inflammatory effects in muscle tissue, it can also be described, for example, as a cell preparation used to suppress inflammation in muscle tissue. Furthermore, the preparation for suppressing muscle mass reduction of the present invention can suppress the induction of expression of inflammatory cytokine genes such as TNF (Tumor Necrosis Factor)-α and / or chemokine genes such as MCP-1 (CCL2, monocyte chemotactic and activating factor) in muscle tissue. Therefore, it can also be described as a cell preparation used to suppress the expression or induction of inflammatory cytokine genes and / or chemokine genes in muscle tissue.

[0033] The mRNA sequences and protein amino acid sequences of human TNF-α and human MCP-1 can be referenced, for example, from sequences registered under the following Genbank accession numbers. Human TNF-α: mRNA accession number: NM_000594 Protein accession number: NP_000585 Human MCP-1 mRNA accession number: NM_002982 Protein accession number: NP_002973

[0034] In the present invention, it is preferable that the cell preparation exhibits a muscle cell proliferation-promoting effect. This proliferation-promoting effect can be evaluated, for example, according to Example 6 described below, using muscle cell proliferation or the expression of muscle cell proliferation-inducing genes such as TGF (Transforming growth factor)-β1 as indicators. Specifically, it can be evaluated using muscle cell proliferation or the expression level of muscle cell proliferation-inducing genes in the muscle tissue of SAMP10 mice (e.g., gastrocnemius or soleus muscle) as indicators (muscle proliferation assay). In the above evaluation, for example, if the number of muscle cells in the group administered the test substance is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to the number of muscle cells in the group not administered the test substance, then the test substance can be evaluated as having a muscle cell proliferation-promoting effect. Furthermore, in the above evaluation, for example, if the expression level of muscle cell proliferation-inducing genes in the group administered the test substance is 1.5 times or more, 2 times or more, 2.5 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, 100 times or more, 25 times or more, 500 times or more, 125 times or more, 250 times or more, or 500 times or more compared to the expression level of muscle cell proliferation-inducing genes in the group not administered the test substance, then the test substance can be evaluated as having a muscle cell proliferation-promoting effect. The cell preparation can increase the number of muscle cells by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, with an upper limit of, for example, 150% or less or 100% or less.The cell preparation can, for example, in the muscle proliferation assay, increase the expression level of the muscle cell proliferation-inducing gene by 1.5 times or more, 2 times or more, 2.5 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, 100 times or more, 125 times or more, 250 times or more, or 500 times or more compared to the group administered the cell preparation, with the upper limit being, for example, 1000 times or less or 750 times or less. The preparation for suppressing the decrease in muscle mass of the present invention can promote the proliferation of the muscle cells (also referred to as "accelerating," "increasing," or "enhancing," hereinafter the same), and therefore can also be used, for example, as a cell preparation for promoting the proliferation of muscle cells or skeletal muscle tissue. Furthermore, since the preparation for suppressing the decrease in muscle mass of the present invention can induce the expression of the muscle cell proliferation-inducing gene, it can also be used, for example, as a cell preparation for inducing (inducing the expression of) muscle tissue proliferation-promoting genes. The muscle cells are, for example, skeletal muscle cells.

[0035] The mRNA base sequence and protein amino acid sequence of human TGF-β1 can be referenced, for example, from sequences registered under the following Genbank accession numbers. Human TGF-β1: mRNA accession number: NM_000660 Protein accession number: NP_000651

[0036] In the present invention, the cell preparation is preferably used to promote the repair of muscle tissue, and more preferably, muscle fibers of skeletal muscle. The muscle tissue repair ability can be evaluated, for example, according to Example 7 described below, using as an indicator the expression level of muscle tissue repair-promoting genes such as the Sirt1 (Sirtuin 1) gene and the myosin heavy chain (MHC) gene, or the expression level of proteins of repair-promoting genes such as Sirt1 and / or myosin heavy chain. Specifically, it can be evaluated using as an indicator the expression level of muscle tissue repair-promoting genes in the muscle tissue of SAMP10 mice (e.g., gastrocnemius or soleus muscle) (muscle repair assay). In the above evaluation, for example, if the expression level of muscle tissue repair-promoting genes in the group administered the test substance is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to the expression level of muscle tissue repair-promoting genes in the group not administered the test substance, then the test substance can be evaluated as having a muscle tissue repair effect. The cell preparation can increase the expression level of muscle tissue repair-promoting genes by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, with an upper limit of, for example, 150% or less or 100% or less. Since the preparation for suppressing muscle mass decline of the present invention exhibits the ability to repair muscle tissue or skeletal muscle, it can also be called, for example, a cell preparation used for the repair of muscle tissue or skeletal muscle. Furthermore, since the preparation for suppressing muscle mass loss of the present invention can induce the expression of repair-promoting genes or the expression of repair-promoting gene proteins in muscle tissue or skeletal muscle, it can also be described as a cell preparation used to promote muscle tissue repair or a cell preparation used to induce the expression of repair-promoting genes in muscle tissue. The muscle tissue is, for example, skeletal muscle tissue.

[0037] The base sequences of human SIRT1 and MHC mRNA, and the amino acid sequences of the proteins can be referenced, for example, from sequences registered with the following Genbank accession numbers. Human SIRT1: mRNA accession number: NM_012238 Protein accession number: NP_036370 Human MHC: mRNA accession number: NM_005963 Protein accession number: NP_005954

[0038] In the present invention, the cell preparation preferably suppresses damage to muscle tissue, more preferably skeletal muscle. The ability to suppress muscle tissue damage can be evaluated, for example, by using the expression level of muscle damage-inducing genes such as the cathepsin K (CatK, Cathepsin K, CTSK) gene as an indicator, according to Example 7 described below. CatK causes muscle damage, for example, by inducing the degradation of muscle cells. Since the preparation for suppressing muscle mass reduction of the present invention exhibits the ability to suppress damage to muscle tissue or skeletal muscle, it can also be described as a cell preparation used to suppress damage to muscle tissue or skeletal muscle. Furthermore, since the preparation for suppressing muscle mass reduction of the present invention can suppress the expression of muscle damage-inducing genes in muscle tissue or skeletal muscle, it can also be described as a cell preparation used to suppress the expression of muscle damage-inducing genes. The muscle tissue is, for example, skeletal muscle.

[0039] The base sequence of human CatK mRNA and the amino acid sequence of the protein can be referenced, for example, from the sequences registered with the following Genbank accession numbers. · Human CatK: mRNA accession number: NM_000396 Protein accession number: NP_000387

[0040] In the present invention, the method for producing (preparing) umbilical cord-derived cells includes, for example, a step of isolating cells from the umbilical cord, and optionally includes a step of subculturing the isolated cells. Specifically, the preparation method includes, for example, (1) a step of cutting the umbilical cord, (2) a step of culturing the umbilical cord section, and (3) a step of subculturing. Another example is the preparation method including, for example, (A) a step of dissociating the tissue by cutting the umbilical cord or treating it with enzymes, or both, (B) a step of culturing the umbilical cord tissue, and (C) a step of subculturing. The umbilical cord-derived cells may be a population of uniform cells or a heterogeneous population of cells.

[0041] When preparing umbilical cord-derived cells by a method including steps (1) to (3) above, or by a method including steps (A) to (C) above, the method can be carried out as follows as an example. However, the method for preparing umbilical cord-derived cells is not limited to the following example.

[0042] First, a method including steps (1) to (3) described above will be explained. Step (1), cutting the umbilical cord, can be carried out, for example, by cutting the umbilical cord obtained by the method described above, including the amniotic membrane, blood vessels, perivascular tissue and / or Wharton's jelly, using mechanical force (shredding force or shearing force). The size of the umbilical cord section obtained by cutting is not particularly limited, for example, 1 to 10 mm. 3 , 1-5mm 3 , 1-4mm 3 , 1-3mm 3 or 1-2mm 3 These are some examples.

[0043] Next, in step (2) culturing the umbilical cord section, for example, the cut umbilical cord section is seeded in an incubator such as a petri dish, dish, or flask, and cultured in a culture medium suitable for umbilical cord-derived cells. In step (2), it is preferable not to treat the umbilical cord section with digestive enzymes.

[0044] In the present invention, the "incubator" may be, for example, an incubator having a solid surface. As the incubator, for example, an incubator used for culturing cells, tissues, and / or organs can be used. The "solid surface" means, for example, any material that enables binding with the umbilical cord-derived cells. Specifically, the material may be, for example, a plastic material that has been treated to promote the binding of mammalian cells to its surface (for example, by increasing its hydrophilicity). The type of culture vessel having a solid surface is not particularly limited and may include, for example, a petri dish, a dish, a flask, etc.

[0045] In the present invention, the "culture medium suitable for umbilical cord-derived cells" can be prepared, for example, by adding an additive such as serum to a basal culture medium. Examples of such additives include serum and / or one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The culture medium may also be further enriched, for example, by adding substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antifungal agents, antioxidants, pyruvate, buffers, and inorganic salts as needed. The basal medium is not particularly limited and includes, for example, Dulbecco's Modified Eagle's Medium (DMEM) (high or low glucose), Modified DMEM, DMEM / MCDB 201, Eagle's Basic Medium, Ham's F10 Medium (F10), Ham's F-12 Medium (F12), Iscove's Modified Dulbecco's Medium (IMDM), Fischer's Medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM / F12, RPMI 1640, CELL-GRO-FREE, and mixed media thereof. The serum includes, for example, animal-derived serum such as human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, and rat serum. The amount of serum added to the basal medium is, for example, 5 v / v% to 15 v / v%, preferably about 10 v / v%. The fatty acids are not particularly limited and include, for example, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. The lipids are not particularly limited and include, for example, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. The amino acids are not particularly limited and include, for example, the L-forms, D-forms, or mixtures (DL-forms) of amino acids such as L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine.The aforementioned proteins are not particularly limited and include, for example, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. The aforementioned polysaccharides are not particularly limited and include, for example, hyaluronic acid and glycosaminoglycans such as heparan sulfate. The aforementioned growth factors are not particularly limited and include, for example, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitor (LIF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and erythropoietin (EPO). The aforementioned antibiotic and / or antifungal agents are not particularly limited and include, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, nystatin, and mixtures thereof.

[0046] In step (2) above, it is preferable to hold down the seeded umbilical cord sections using a plate or the like during the culture period in order to prevent them from floating in the culture medium. For example, the plate described in Japanese Patent Application Publication No. 2015-70824 can be used as an example of such a plate.

[0047] In step (2) above, the culture conditions are not particularly limited and general culture conditions for cells, tissues, organs, etc. can be used. Specifically, the CO2 concentration in step (2) above is, for example, 0 to 5%. The O2 concentration in step (2) above is, for example, 2 to 25%, preferably 5 to 20%. The culture temperature in step (2) above is, for example, 25 to 40°C, preferably about 37°C (35 to 39°C).

[0048] In step (2) above, the culture period is not particularly limited, but it is preferable to culture the cells until they migrate from the umbilical cord section and the cells reach a confluence of 50%, 60%, 70%, 80% or more of the culture vessel.

[0049] And in the step (2), for example, after the culture, in order to remove unbound cells and cell fragments, the cells are washed and detached with a detachment agent containing a chelating agent such as EDTA, a protease such as trypsin, collagenase, dispase, a glycosidase such as hyaluronidase, or a mixture thereof. And in the step (2), for example, the detachment solution containing the cells and the umbilical cord sections can be filtered using a cell strainer or the like to obtain only the cells as umbilical cord-derived cells. The obtained umbilical cord-derived cells can be seeded, for example, into the aforementioned incubator and cultured using the aforementioned culture medium.

[0050] In the step (3), by subculturing, the umbilical cord-derived cells can be appropriately proliferated to the required number. In the step (3), in the subculture, the cells detached by the detachment agent may be seeded at an appropriate cell density into a separately prepared incubator and the culture may be continued. The cell density (seeding density) when seeding the cells is, for example, 1×10 2 ~1×10 5 cells / cm 2 、5×10 2 ~5×10 4 cells / cm 2 、1×10 3 ~1×10 4 cells / cm 2 、2×10 3 ~1×10 4 cells / cm 2 etc., and preferably, 2×10 3 ~1×10 4 cells / cm 2 is used. The seeding density is preferably adjusted, for example, so that the period until reaching an appropriate confluence is 3 to 7 days. In the subculture of the step (3), the medium may be appropriately changed as necessary.

[0051] The number of subcultures in the step (3) is not particularly limited, and may be performed, for example, until senescence where cell division stops. The number of subcultures in the step (3) is preferably, for example, subcultured 3 to 25 times, and more preferably subcultured 4 to 12 times, from the viewpoint of using for treatment.

[0052] Next, a method including steps (A) to (C) described above will be explained. Step (A), the enzymatic treatment step, can be carried out by enzymatic treatment of the umbilical cord obtained by the method described above, including the amniotic membrane, blood vessels, perivascular tissue and / or Wharton's jelly, to dissociate the tissue. The enzyme used in the enzymatic treatment is not particularly limited and may include, for example, proteases such as collagenase and dispase; glycosyltransferases such as hyaluronidase; and the like.

[0053] Next, the steps of (B) culturing the umbilical cord tissue and (C) subculturing can be carried out, for example, in the same manner as the steps of (2) culturing the umbilical cord tissue and (3) subculturing, respectively.

[0054] This allows the umbilical cord-derived cells to be obtained after step (3) or (C).

[0055] The cells obtained by the umbilical cord-derived cell preparation method described above may be analyzed using conventional methods such as flow cytometry for surface antigens, etc., to confirm that they are umbilical cord-derived cells. Alternatively, the cells obtained by the umbilical cord-derived cell preparation method may be evaluated as umbilical cord-derived cells by measuring the amount of various proteins produced by the cells.

[0056] The cells obtained by the umbilical cord-derived cell preparation method described above may be prepared for therapeutic use as is, or they may be cryopreserved. Cryopreservation is carried out, for example, by suspending the cells in a cryopreservation solution capable of preserving umbilical cord-derived cells and storing them at -80°C to -180°C. The cryopreservation solution is not particularly limited and may include, for example, an aqueous solution containing a cryoprotectant and glucose. Examples of the cryoprotectant include dimethyl sulfoxide (hereinafter also referred to as "DMSO"), dextran, glycerol, propylene glycol, and 1-methyl-2-pyrrolidone, and preferably DMSO and / or propylene glycol, more preferably DMSO. The cryoprotectant is present in the cryopreservation solution in an amount of, for example, 1 to 15 w / v%, preferably 5 to 15 w / v%, more preferably 5 to 12 w / v%, and even more preferably 8 to 11 w / v%. The freeze-drying protection agent is, for example, present in the freeze-preservation solution at a concentration of 1 to 15 v / v%, preferably 5 to 15 v / v%, more preferably 5 to 12 v / v%, and even more preferably 8 to 11 v / v%.

[0057] The glucose contained in the cryopreservation solution is, for example, present in the cryopreservation solution at a concentration of 0.5 to 10 w / v%, preferably 1 to 10 w / v%, more preferably 2 to 8 w / v%, and even more preferably 2 to 5 w / v%.

[0058] The cryopreservation solution may further contain other components. Examples of other components include pH adjusters and thickeners. Examples of pH adjusters include sodium bicarbonate, HEPES, and phosphate buffer. If phosphate buffer is not added to the basic Stock Solution (BSS), a pH adjuster containing sodium chloride, which has the function of providing buffering capacity at a pH near that suitable for the umbilical cord-derived cells, may also be used. It is preferable to use phosphate buffer as the pH adjuster. It is preferable to use the pH adjuster to adjust the pH of the cryopreservation solution to, for example, about 6.5 to 9, preferably 7 to 8.5. In the present invention, "phosphate buffer" refers to a buffer containing, for example, sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate (anhydrous), potassium chloride, and potassium dihydrogen phosphate (anhydrous), and a buffer containing sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous) is particularly preferred. The pH adjusting agent is, for example, present in the cryopreservation solution at a concentration of 0.01 to 1 w / v%, preferably 0.05 to 0.5 w / v%.

[0059] The cryopreservation solution may or may not contain natural animal-derived components. Examples of such natural animal-derived components include the aforementioned serum and basal culture medium. It is preferable that the cryopreservation solution does not contain natural animal-derived components. A cryopreservation solution that does not contain natural animal-derived components avoids the problem of quality differences between lots of natural animal-derived components, suppresses the possibility of changes in the properties of cells in umbilical cord tissue due to various cytokines, growth factors, and hormones contained in serum, and further suppresses the influence of components of unknown origin contained in the basal culture medium. For this reason, a cryopreservation solution that does not contain natural animal-derived components is particularly useful in clinical use.

[0060] The cryopreservation solution may further contain a thickening agent. The thickening agent is not particularly limited and can be any agent capable of forming a cryopreservation solution that can adequately preserve the umbilical cord tissue. Examples of the thickening agent include carboxymethylcellulose (hereinafter also referred to as "CMC"), sodium carboxymethylcellulose (hereinafter also referred to as "CMC-Na"), organic acid polymers, propylene glycol alginate, sodium alginate, etc. CMC and CMC-Na are preferred as thickening agents, with CMC-Na being particularly preferred. Sodium polyacrylate is preferred as the organic acid polymer. The thickening agent is present in the cryopreservation solution in an amount of 0.1 to 1 w / v%, preferably 0.1 to 0.5 w / v%, and more preferably 0.2 to 0.4 w / v%, for example.

[0061] The cryopreservation solution is preferably an aqueous solution. The osmotic pressure of the cryopreservation solution is preferably 1000 mOsm or more, and more preferably 1000 to 2700 mOsm, in order to maintain its performance as a preservation solution.

[0062] The cryopreservation solution is preferably an aqueous solution containing a thickener, a freeze-thaw agent, and glucose, and free from natural animal-derived components. More preferably, the cryopreservation solution is an aqueous solution containing CMC-Na, DMSO, and glucose, and free from natural animal-derived components. Even more preferably, the cryopreservation solution is an aqueous solution containing 0.1 to 1 w / v% CMC-Na, 1.0 to 15 w / v% DMSO, and 0.5 to 10 w / v% glucose, and free from natural animal-derived components.

[0063] The cells obtained by the umbilical cord-derived cell preparation method described above may be used as cell preparations for various purposes by, for example, mixing them with an intravenous fluid preparation. Furthermore, when the umbilical cord-derived cells are cryopreserved, the cryopreserved umbilical cord-derived cells may be suspended in the cryopreservation solution and used directly as cell preparations for various purposes after thawing, or they may be mixed with an intravenous fluid preparation after thawing, and the resulting mixture may be used as cell preparations for various purposes. When mixing with an intravenous fluid preparation, the culture medium or cryopreservation solution in which the umbilical cord-derived cells are suspended may be mixed with the intravenous fluid preparation, or the cells may be separated from the solvent by centrifugation or the like in the culture medium or cryopreservation solution, and only the cells may be mixed with the intravenous fluid preparation. In the preparation method described above, for example, in order to avoid the complexity of the procedure, it is preferable not to include a step of culturing the frozen cells after thawing, or to directly mix the cryopreservation solution in which the thawed cells are suspended with the intravenous fluid preparation.

[0064] In the present invention, the "infusion preparation" refers to solutions such as infusions used in the treatment of humans, and specific examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, Solution No. 1, Solution No. 2, Solution No. 3, Solution No. 4, etc.

[0065] The preparation for suppressing muscle mass loss according to the present invention may be a kit containing the umbilical cord-derived cells in addition to the infusion solution.

[0066] The preparation for inhibiting muscle mass loss of the present invention may contain, in addition to or in lieu of, the aforementioned intravenous solution preparation, a pharmaceutically acceptable carrier. Examples of such carriers include suspensions for administering the cell preparation, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, media, pH adjusters, analgesics, buffers, sulfur-containing reducing agents, antioxidants, etc., and can be appropriately added within a range that does not impede the effects of the present invention.

[0067] The suspending agent is not particularly limited and includes, for example, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0068] The aforementioned solution additive is not particularly limited and includes, for example, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0069] The stabilizer is not particularly limited and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, and the like.

[0070] The isotonic agent is not particularly limited and examples include D-mannitol and sorbitol.

[0071] The preservative is not particularly limited and examples include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, and the like.

[0072] The adsorption inhibitor is not particularly limited and includes, for example, human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, hydrogenated castor oil, polyethylene glycol, and the like.

[0073] The sulfur-containing reducing agent is not particularly limited and can be, for example, N-acetylcysteine, N-acetylhomocysteine, thioxytoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, thioalkanoic acid having 1 to 7 carbon atoms, or any other substance having a sulfohydryl group.

[0074] The antioxidant is not particularly limited and examples include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, or chelating agents such as ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0075] The preparation for suppressing muscle mass loss of the present invention may further contain, as appropriate, commonly added components such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts such as sodium citrate, potassium citrate, and sodium acetate; and sugars such as glucose. In addition, as an anticoagulant and / or pH adjuster, for example, ACD-A solution (composed of sodium citrate hydrate, citric acid hydrate, glucose, etc.) may be added.

[0076] The preparation for suppressing muscle mass loss of the present invention may be mixed with organic substances such as biopolymers; inorganic substances such as hydroxyapatite; etc., for topical administration. Specifically, it may be mixed with collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer and its chemical derivatives.

[0077] The preparation for suppressing muscle mass loss according to the present invention is, for example, in via You can use it as well, in vivo It may also be used in the following ways. The preparation for inhibiting muscle mass decline of the present invention can be used, for example, as a research reagent or as a pharmaceutical. In the latter case, the preparation for inhibiting muscle mass decline of the present invention can also be called a cell preparation or pharmaceutical cell preparation used in the treatment of age-related muscle loss.

[0078] The subjects to whom the preparation for suppressing muscle mass reduction of the present invention is administered are not particularly limited. in vivo When used in the present invention, the target of administration may be, for example, humans or non-human animals other than humans. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions; birds; fish; etc. The preparation for suppressing the decrease in muscle mass of the present invention may be used in the present invention. in via When used in this manner, the target of administration may include, for example, cells, tissues, organs, etc., and the cells may include, for example, cells collected from a living organism, cultured cells, etc., and the tissues or organs may include, for example, tissues (living tissues) or organs collected from a living organism. The cells may include, for example, muscle cells, iPS cells (induced pluripotent stem cells), stem cells, etc.

[0079] The preparation for suppressing the decrease in muscle mass of the present invention in vivo When used in this manner, the subjects of administration may be subjects in which inflammation occurs in muscle tissue, subjects in which the expression of inflammatory cytokine genes is induced (also called "enhancement," "increase," "improvement," or "enhancement," hereinafter the same) and / or the expression of chemokine genes is induced in muscle tissue, subjects in which interstitial fibrosis is induced or occurring in muscle tissue, subjects in which the expression of muscle tissue repair-promoting genes is suppressed, subjects in which mitochondrial dysfunction occurs in muscle cells, subjects in which the number of mitochondria in muscle cells is reduced, subjects in which damage occurs in muscle tissue, subjects in which the expression of muscle tissue damage-inducing genes is induced, and / or subjects in which apoptosis in muscle cells is improved (enhanced).

[0080] The preparation for suppressing the decrease in muscle mass of the present invention in vivoWhen used in the present invention, the target recipient is preferably one who is experiencing a decrease in muscle mass. Furthermore, in the preparation for suppressing muscle mass loss of the present invention, the target recipient is preferably one who is experiencing a decrease in muscle mass due to aging, as it can suppress the decrease in muscle mass in age-related muscle loss, as will be described later. Age-related muscle loss occurs, for example, in middle-aged and elderly people. For this reason, the target recipient is preferably middle-aged and elderly people. Examples of middle-aged and elderly people include people aged 40 or older, 50 or older, 60 or older, 70 or older, or 80 or older.

[0081] The conditions for use (administration conditions) of the preparation for suppressing muscle mass loss of the present invention are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of target to be administered to.

[0082] Methods for administering the preparation for suppressing muscle mass loss of the present invention include, for example, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, and intravenous administration. However, intravenous administration is preferred because it can be administered safely and stably regardless of the administerer's skill.

[0083] The dosage of the preparation for inhibiting muscle mass loss of the present invention is the amount of cells that, when administered to a subject, will produce a muscle mass loss inhibiting effect (therapeutic effect) against the disease compared to a subject that does not receive the preparation. Specifically, the dosage can be appropriately determined, for example, by the subject's age, weight, symptoms, etc. As a specific example, the dosage may be, for example, 10 umbilical cord-derived cells per administration. 4 ~10 9 pieces / kg weight, 10 4 ~10 8 pieces / kg weight, 10 4 ~10 7 The number of units / kg body weight can be increased, preferably 10 4 ~10 8 pieces / kg weight, 10 4 ~10 7 The number of cells per kg of body weight is 10 cells per dose. The dose is, for example, 10 cells per dose, based on the number of umbilical cord-derived cells. 4 ~10 9 pieces, 10 6 ~109 pieces, 10 4 ~10 8 pieces, 10 4 ~10 7 The number of items is listed, preferably 10. 4 ~10 8 pieces, 10 4 ~10 7 The number of cells is one. The dose can also be, for example, the number of umbilical cord-derived cells in the cell preparation.

[0084] The preparation for suppressing muscle mass loss of the present invention is administered once or multiple times. The multiple times may be, for example, two, three, four, five, or more times. The number of administrations may be determined appropriately while confirming the therapeutic effect on the target. When multiple administrations are performed, the administration interval can be determined appropriately while confirming the therapeutic effect on the target, and examples include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0085] The preparation for inhibiting muscle mass loss of the present invention may be used in combination, for example, with other drugs and / or methods used to inhibit muscle mass loss. Examples of methods used to inhibit muscle mass loss include exercise therapy such as resistance exercise, and specific examples can be found in the following references 1 to 3. Reference 1: Hidetaka Wakabayashi, "Practical Aspects of Exercise Therapy for Sarcopenia," 2013, Nihon Iji Shinpo, No. 4677, pp. 32-36. Reference 2: Hidenori Arai et.al., “Special Issue: Clinical Guidelines for Sarcopenia. Guest Editor: Hidenori Arai. This publication has been supported by The Japanese Association on Sarcopenia and Frailty, The Japan Geriatrics Society and National Center for Geriatrics and Gerontology (NCGG) (Japan) Chapter 4 Treatment of sarcopenia”, 2018, Geriatrics & Gerontology International, pages 28-44 Reference 3: Masafumi Kuzuya et.al., “Special Issue: Clinical Guide for Frailty. Guest Editors: Shosuke Satake and Hidenori Arai. This publication has been supported by The Japanese Association on Sarcopenia and Frailty, The Japan Geriatrics Society and National Center for Geriatrics and Gerontology (NCGG) (Japan) Chapter 3 Frailty prevention”, 2020, Geriatrics & Gerontology International, pages 20-24

[0086] The preparation for suppressing muscle mass decline of the present invention can be suitably used in subjects experiencing muscle mass decline, and specifically in subjects with age-related muscle loss. Age-related muscle loss refers to symptoms or diseases in which muscle mass, such as skeletal muscle, decreases with age. Examples of age-related muscle loss include sarcopenia and frailty.

[0087] As described above, the preparation for suppressing muscle mass loss of the present invention can suppress muscle mass loss in a subject. For this reason, the present invention may also include a method for suppressing muscle mass loss in a subject. In this case, the present invention is a method for suppressing muscle mass loss in a subject, wherein the subject is given the cell preparation used for suppressing muscle mass loss of the present invention. The method for suppressing muscle mass loss of the present invention includes, for example, the step of administering the cell preparation used for suppressing muscle mass loss of the present invention to the subject. The administration conditions in the administration step can be described by referring to the above explanation.

[0088] As described above, the preparation for suppressing muscle mass loss of the present invention can suppress muscle mass loss in a subject. Therefore, the present invention is a method for treating a subject experiencing muscle mass loss, wherein the subject is given the cell preparation used for suppressing muscle mass loss of the present invention. The method for treating a subject of the present invention includes, for example, the step of administering the cell preparation used for suppressing muscle mass loss of the present invention to the subject. The administration conditions in the administration step can be described by referring to the above explanation.

[0089] As described above, the preparation for suppressing muscle mass loss of the present invention can suppress muscle mass loss in a subject, and therefore can treat, for example, age-related muscle loss. For this reason, the present invention relates to a method for treating a patient with age-related muscle loss, wherein the patient with age-related muscle loss is administered the cell preparation used for suppressing muscle mass loss of the present invention. The administration conditions in the administration step can be described in the above explanation.

[0090] In the present invention, "treatment" may be used in any of the following senses: suppression, prevention, inhibition, or delay of the onset of the target disease; cessation, suppression, inhibition, or delay of the progression of the target disease or its symptoms once it has developed; and improvement or remission of the target disease.

[0091] <Uses of cell preparations> In another embodiment, the present invention provides a cell preparation or a method for improving mitochondrial function in muscle tissue. In this case, the present invention is a cell preparation used for improving mitochondrial function in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for improving mitochondrial function in target muscle tissue, wherein the cell preparation used for improving mitochondrial function in muscle tissue according to the present invention is used as the target. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass decline according to the present invention.

[0092] In another embodiment, the present invention provides a cell preparation for use in increasing the number of mitochondria in muscle tissue or a method for increasing the number of mitochondria in muscle tissue. In this case, the present invention is a cell preparation for use in increasing the number of mitochondria in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for increasing the number of mitochondria in target muscle tissue, wherein the target is a cell preparation for use in increasing the number of mitochondria in muscle tissue. The present invention can draw upon the description of the cell preparation for use in suppressing the decrease in muscle mass of the present invention.

[0093] In another embodiment, the present invention provides a cell preparation or a method for inducing the expression of mitochondrial function-enhancing genes in muscle tissue. In this case, the present invention is a cell preparation used for inducing the expression of mitochondrial function-enhancing genes in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for inducing the expression of mitochondrial function-enhancing genes in target muscle tissue, wherein the target is a cell preparation used for inducing the expression of mitochondrial function-enhancing genes in muscle tissue. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass loss in the present invention.

[0094] In another embodiment, the present invention provides a cell preparation or a method for inhibiting apoptosis of muscle cells. In this case, the present invention is a cell preparation used for inhibiting apoptosis of muscle cells, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for inhibiting apoptosis in target muscle cells, wherein the cell preparation used for inhibiting apoptosis of muscle cells is used as the target. The present invention can draw upon the description of the cell preparation used for inhibiting the decrease in muscle mass of the present invention.

[0095] In another embodiment, the present invention provides a cell preparation for use in suppressing inflammation in muscle tissue or a method for suppressing inflammation in muscle tissue. In this case, the present invention is a cell preparation for use in anti-inflammatory (inflammation suppression) purposes in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention is a method for suppressing inflammation in target muscle tissue, wherein the target is a cell preparation for use in suppressing inflammation in muscle tissue. The present invention can draw upon the description of the cell preparation for use in suppressing muscle mass reduction of the present invention.

[0096] In another embodiment, the present invention provides a cell preparation for use in suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, or a method for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue. In this case, the present invention is a cell preparation for use in suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in target muscle tissue, wherein the target is a cell preparation for use in suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue. The present invention can draw upon the description of the cell preparation for use in suppressing the decrease in muscle mass of the present invention.

[0097] In another embodiment, the present invention provides a cell preparation or a method for promoting the proliferation of muscle cells. In this case, the present invention is a cell preparation used for promoting the proliferation of muscle cells, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for promoting the proliferation of target muscle cells, wherein the target is a cell preparation used for promoting the proliferation of muscle cells. The present invention can draw upon the description of the cell preparation used for suppressing the decrease in muscle mass of the present invention.

[0098] In another embodiment, the present invention provides a cell preparation or a method for inducing the expression of a gene that promotes muscle cell proliferation for use in inducing the expression of a gene that promotes muscle cell proliferation. In this case, the present invention is a cell preparation used for inducing the expression of a gene that promotes muscle cell proliferation, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for inducing the expression of a gene that promotes muscle cell proliferation, The target is a cell preparation used to induce the expression of gene that promotes muscle cell proliferation. The present invention can be described by reference to the above description of the cell preparation used to suppress the decrease in muscle mass.

[0099] In another embodiment, the present invention provides a cell preparation or a method for repairing muscle tissue. In this case, the present invention is a cell preparation for repairing muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention is a method for repairing target muscle tissue, wherein the target is a cell preparation for repairing muscle tissue. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass loss in the present invention.

[0100] In another embodiment, the present invention provides a cell preparation or a method for inducing the expression of muscle tissue repair genes for use in inducing the expression of muscle tissue repair genes. In this case, the present invention is a cell preparation for use in inducing the expression of muscle tissue repair genes, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for inducing the expression of repair genes in target muscle tissue, wherein the target is a cell preparation for use in inducing the expression of muscle tissue repair genes. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass loss in the present invention.

[0101] In another embodiment, the present invention provides a cell preparation or a method for suppressing muscle tissue damage. In this case, the present invention is a cell preparation used for suppressing muscle tissue damage, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for suppressing damage to target muscle tissue, wherein the target is a cell preparation used for suppressing muscle tissue damage. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass reduction in the present invention.

[0102] In another embodiment, the present invention provides a cell preparation or a method for suppressing the expression of damage-inducing genes in muscle tissue. In this case, the present invention is a cell preparation used for suppressing the expression of damage-inducing genes in muscle tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for suppressing the expression of damage-inducing genes in target muscle tissue, wherein the target is a cell preparation used for suppressing the expression of damage-inducing genes in muscle tissue. The present invention can draw upon the description of the cell preparation used for suppressing muscle mass reduction in the present invention.

[0103] In another embodiment, the present invention provides a cell preparation or a method for inhibiting fibrosis of muscle tissue interstitial tissue for use in inhibiting fibrosis of muscle tissue interstitial tissue. In this case, the present invention is a cell preparation for use in inhibiting fibrosis of muscle tissue interstitial tissue, wherein the cell preparation includes umbilical cord-derived cells. The present invention also provides a method for inhibiting fibrosis of muscle tissue interstitial tissue for use in the target, wherein the cell preparation for use in inhibiting fibrosis of muscle tissue interstitial tissue is used as the target. The present invention can draw upon the description of the cell preparation used for inhibiting muscle mass reduction in the present invention.

[0104] <Use of cell preparations> The present invention relates to a cell composition for use in suppressing the decline in muscle mass, treating age-related muscle loss, improving mitochondrial function in muscle tissue, increasing the number of mitochondria in muscle tissue, inducing the expression of genes that improve mitochondrial function in muscle tissue, suppressing apoptosis in muscle cells, suppressing inflammation in muscle tissue, suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, promoting the proliferation of muscle cells, inducing the expression of genes that promote the proliferation of muscle cells, repairing muscle tissue, inducing the expression of genes that repair muscle tissue, suppressing muscle tissue damage, and / or suppressing the expression of genes that induce muscle tissue damage, wherein the cell preparation includes umbilical cord-derived cells. The present invention relates to the use of umbilical cord-derived cells for producing cell compositions for use in suppressing muscle mass decline, treating age-related muscle loss, improving mitochondrial function in muscle tissue, increasing the number of mitochondria in muscle tissue, inducing the expression of mitochondrial function-enhancing genes in muscle tissue, suppressing apoptosis in muscle cells, suppressing inflammation in muscle tissue, suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, promoting muscle cell proliferation, inducing the expression of muscle cell proliferation-promoting genes, repairing muscle tissue, inducing the expression of muscle tissue repair genes, suppressing muscle tissue damage, and / or suppressing the expression of muscle tissue damage-inducing genes. The present invention can be further described by reference to the description of the preparation for suppressing muscle mass decline of the present invention. [Examples]

[0105] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to their respective protocols.

[0106] [Example 1] We have confirmed that sarcopenia can be treated with the cell preparations of the present invention.

[0107] (1) Preparation of umbilical cord-derived cells Umbilical cord-derived cells were collected using the method described in Cytotherapy, 18, 229-241, 2016. Specifically, all tissue elements of the umbilical cord (including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly) were collected with the consent of the donor and approval from the Ethics Committee of the Institute of Medical Science, University of Tokyo, in 1-2 mm increments. 3 The umbilical cord was shredded into fragments and seeded onto a culture dish. Then, using an improved Explant method, cells were obtained by covering them with Cell Amigo (manufactured by Tsubakimoto Chain Co., Ltd.) and culturing them in α-minimal essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. These cells exhibited plastic adhesion.

[0108] (2) Confirmation of surface antigens Next, the presence or absence of surface antigen expression was checked in the obtained umbilical cord-derived cells. The presence or absence of surface antigen expression was confirmed by FACS analysis using specific antibodies against each antigen. As for surface antigens of umbilical cord-derived cells, CD73, CD105, CD90, CD44, and HLA-class I were positive, while HLA-class II, CD34, CD45, CD19, CD80, CD86, CD40, and CD11b were negative. Furthermore, HLA-G5 and PD-L2 were positive. In addition, HLA-G was weakly positive, CD49d (ITGA4) and CD184 (CXCR4) were negative to weakly positive, and CD29 (ITGB1) was positive.

[0109] Furthermore, it was confirmed by Realtime PCR that the umbilical cord-derived cells highly express the HGF (Hepatic Growth Factor) gene under normal conditions, and that the IDO (Indoleamine 2,3-dioxygenase) gene expression is induced under inflammatory conditions (IFN-γ 100 ng / ml). In particular, HGF expression was found to be higher in umbilical cord-derived cells than in bone marrow-derived mesenchymal stem cells. In addition, it was confirmed by ELISA that co-culturing umbilical cord-derived cells with MLR (mixed allogeneic lymphocyte reaction) induces PGE2 secretion.

[0110] (3) Preparation of cell preparations The obtained umbilical cord-derived cells were seeded in Corning® CellBIND® surface 100 mm dishes (Corning, Product Number: #3292) and subcultured from Passage 1 to Passage 4 to proliferate. The culture medium used during the subculture was CiMS®-BM (Nipro Corporation, Product Code: 87-070), a basic culture medium for human mesenchymal stem cells, to which CiMS®-sAF (Nipro Corporation, Product Code: 87-072), an animal-free additive for basic culture medium for human mesenchymal stem cells, was added in the proportion specified in the package insert. Subsequently, the cells were detached using TrypLE® Select Enzyme (1X), no phenol red (ThermoFisher, Product Number: 12563011) and 1 × 10⁶ cells were removed. 6 The cell preparation was prepared to a concentration of 150 μl per cell, and this was the cell preparation of the present invention.

[0111] (4) Model animals As the animal used in the study, we used SAMP10 (purchased from Chubu Kagaku Shizai Co., Ltd.), a SAMP (Senescence-Accelerated Mouse Prone) strain mouse exhibiting accelerated aging and a short lifespan. SAMP10 is a strain that exhibits traits such as early onset of age-related disorders, including learning and memory impairment, emotional disorders, and senescent amyloidosis, accompanied by brain atrophy. SAMP10 has a short lifespan of about one year, and aging phenomena can be observed from around 24 weeks of age, so it was used as a model animal for sarcopenia.

[0112] (5) Preparation of mice SAMP10 mice were randomly selected at 24 weeks of age, and the cells were measured using a BD Lodose® 30G insulin subcutaneous injection syringe (Becton Dickinson, catalog number: 326638) until the cells reached 1 × 10⁶ 6 The cells were administered via the tail vein of mice to create the cell / mouse mixture, which served as the mice used in the example. The control group consisted of 24-week-old SAMP10 mice administered the same volume of saline or culture medium.

[0113] Furthermore, the mice from the above-described example and the control mice were each divided into a training group (Ex) and a non-training group. Specifically, the group that received cells but no training (Cell) was designated Example A (n=8), and the group that received cells and training (Cell+Ex) was designated Example B (n=8). The group that received neither cells nor training was designated as the control (n=7), and the group that received no cells but training (Ex) was designated as the reference example (n=7). The training consisted of treadmill training three times a week. The treadmill training involved forcing the mice to run using a training device (mouse treadmill, manufactured by Melquest Co., Ltd.) equipped with a belt conveyor and a device that generates electrical stimulation. The training content was also changed according to the age of the mice. Specifically, for mice aged 24 to 26 weeks, the apparatus was tilted to 0°, followed by a 5-minute warm-up at a speed of 7 m / min, then a 35-minute exercise at a speed of 17 m / min, and finally a 5-minute cool-down at a speed of 7 m / min. For mice aged 27 to 36 weeks, the apparatus was tilted to 5°, followed by a 5-minute warm-up at a speed of 10 m / min, then a 35-minute exercise at a speed of 18 m / min, and finally a 5-minute cool-down at a speed of 10 m / min.

[0114] (6) Results The body weight of each group of mice was measured at 24 weeks, 28 weeks, 32 weeks, and 36 weeks of age. These results are shown in Figure 1. Figure 1 is a graph showing the changes in body weight. In each graph in Figure 1, the vertical axis represents the average body weight (g) of the mice in each group, and the horizontal axis represents the age of the mice in weeks. Figure 1(A) is a graph comparing the changes in body weight of the mice in each group, where the circle (●) plot represents the control group, the square (□) plot represents Example A (Cell), the triangle (▲) plot represents the reference example (Ex), and the triangle (▽) plot represents Example B (Ex + Cell). Furthermore, Figure 1(B) is a graph showing the body weight of the control mice, Figure 1(C) is a graph showing the body weight of the mice in Example A (Cell), Figure 1(D) is a graph showing the body weight of the reference example (Ex), and Figure 1(E) is a graph showing the body weight of the mice in Example B (Ex + Cell). As shown in Figure 1, no significant difference in body weight was observed among the mice in each group.

[0115] Furthermore, the grip strength of each group of mice was measured at 24 weeks, 28 weeks, 32 weeks, and 26 weeks of age. Grip strength was measured using a commercially available small animal grip strength measuring device (mouse grip strength meter, manufactured by AMETEK Chatillon) as follows: First, the mouse was placed on the mesh part of the device and allowed to grasp the mesh with all four limbs. From this position, the mouse's tail was held and pulled horizontally to the ground, and the force (load) required for the mouse to release the mesh was measured. The force just before the mouse released the mesh was defined as the grip strength. These results are shown in Figure 2.

[0116] Figure 2 is a graph showing the change in grip strength per unit of body weight. In each graph in Figure 2, the vertical axis shows the average grip strength per unit of body weight (grip strength (g) / body weight (g)) for each group of mice, and the horizontal axis shows the age of the mice in weeks. Figure 2(A) is a graph comparing the grip strength per unit of body weight (grip strength (g) / body weight (g)) for each group of mice, where the circle (●) plots represent the control group, the square (□) plots represent Example A (Cell), the triangle (▲) plots represent the reference example (Ex), and the triangle (▽) plots represent Example B (Ex + Cell). Figure 2(B) is a graph showing the grip strength of the control mice, Figure 2(C) is a graph showing the grip strength of the mice in Example A (Cell), Figure 2(D) is a graph showing the grip strength of the mice in Reference Example (Ex), and Figure 2(E) is a graph showing the grip strength of the mice in Example B (Ex + Cell).

[0117] As shown in Figure 2, the mice in Example A showed significantly improved grip strength at 32 and 36 weeks of age compared to the control mice. Similarly, the mice in Example B showed significantly improved grip strength at 32 and 36 weeks of age compared to the mice in Reference Example (Ex) and the control mice. Therefore, it was found that the cell preparation of the present invention exhibits a grip strength-improving effect, i.e., a therapeutic effect against muscle weakness in age-related muscle loss.

[0118] Furthermore, endurance was measured for each group of mice at 24 weeks, 28 weeks, 32 weeks, and 36 weeks of age. Endurance was measured using the following endurance measurement method. These results are shown in Figure 3.

[0119] (How to measure endurance) speed slope time Start 6m / min 0° 0~ 5min 6 m / min, 10°, 5-7 minutes 8 m / min 10° 7~9 min 10m / min 10° 9~11min 12m / min 10° 11~13min 14 m / min 10° 13-15 minutes 16m / min 10° 15~17min 18m / min 10° 17~19min 20m / min 10° 19~21min 21m / min 10° 21 min~ First, each mouse was made to run for 5 minutes at a speed of 6 m / min with the device tilted at 0°. After 5 minutes, the tilt was changed to 10°, and the mice were made to run for 2 minutes at a speed of 6 m / min with a device tilt of 10°. Thereafter, the speed was increased by 2 m / min every 2 minutes. The maximum speed was set to 21 m / min, and the time it took for each mouse to become unable to run was measured.

[0120] Figure 3 is a graph showing endurance. In each graph in Figure 3, the vertical axis shows the average endurance (min) of the mice in each group, and the horizontal axis shows the age of the mice in weeks. Figure 3(A) is a graph comparing the endurance (min) of the mice in each group, where the circle (●) plots represent the control, the square (□) plots represent Example A (Cell), the triangle (▲) plots represent the reference example (Ex), and the triangle (▽) plots represent Example B (Ex + Cell). Figure 3(B) is a graph showing the endurance of the control mice, Figure 3(C) is a graph showing the endurance of the mice in Example A (Cell), Figure 3(D) is a graph showing the endurance of the mice in the reference example (Ex), and Figure 3(E) is a graph showing the endurance of the mice in Example B (Ex + Cell).

[0121] As shown in Figure 3, the mice in Example A showed significantly improved endurance at 32 weeks of age compared to the control mice, and further improved endurance at 36 weeks of age. Similarly, the mice in Example B showed significantly improved endurance at 32 weeks of age compared to the reference example (Ex) mice and the control mice, and further improved endurance at 36 weeks of age. Therefore, it was found that the cell preparation of the present invention has an effect of improving endurance, that is, it has a therapeutic effect on muscle weakness in age-related muscle loss.

[0122] [Example 2] We confirmed that the administration of the cell preparation of the present invention improved muscle fiber size.

[0123] Each mouse used in Example 1 was dissected, and the gastrocnemius muscle and soleus muscle were collected, weighed, and observed morphologically. These results are shown in Figure 4.

[0124] Figure 4 is a graph showing the results of muscle weight measurement. Figure 4(A) is a graph showing the weight of the gastrocnemius muscle of each mouse, and Figure 4(B) is a graph showing the weight of the soleus muscle of each mouse. In Figure 4, the vertical axis shows the relative value of muscle mass per unit body weight of the mouse (muscle mass (mg) / body weight (g)), and the horizontal axis shows the type of mouse. As shown in Figure 4, the mice in Example A showed a significant increase in the weight of both the gastrocnemius and soleus muscles 12 weeks after cell administration compared to the control mice. The mice in Example B also showed a significant increase in the weight of both the gastrocnemius and soleus muscles 12 weeks after cell administration compared to the control mice.

[0125] Next, each collected muscle tissue sample was stained with hematoxylin-eosin (HE) and morphologically observed. Furthermore, each collected muscle tissue sample was stained with Masson trichome (MT) to assess the degree of interstitial fibrosis in each sample. These results are shown in Figure 5.

[0126] Figure 5 shows photographs of muscle tissue staining results. In Figure 5, (A) and (B) show the results for the gastrocnemius muscle, with (A) being the result of HE staining and (B) being the result of MT staining. In Figure 5, (C) and (D) show the results for the soleus muscle, with (C) being the result of HE staining and (D) being the result of MT staining. In Figure 5 (B) and (D), the areas stained blue by MT staining, i.e., the areas where the interstitial tissue has become fibrous, are indicated by dashed lines. As shown in Figure 5, compared to control mice, the mice of Example A showed an increase in muscle fiber size in the gastrocnemius and soleus muscles 12 weeks after cell administration. Compared to control mice, the mice of Example B also showed an increase in muscle fiber size in the gastrocnemius and soleus muscles 12 weeks after cell administration. Furthermore, the mice in Examples A and B showed a reduction in interstitial fibrosis in both the gastrocnemius and soleus muscles compared to the control mice. It is known that patients with age-related muscle loss, such as sarcopenia and frailty, experience a decrease in muscle cross-sectional area (muscle fiber size) and interstitial fibrosis. The cell preparations of the present invention can improve muscle cross-sectional area and suppress interstitial fibrosis, and are therefore presumed to have a therapeutic effect on age-related muscle loss.

[0127] From the above, it was found that the cell preparation of the present invention increases muscle mass by improving muscle cross-sectional area and suppressing interstitial fibrosis, thereby exhibiting a therapeutic effect against age-related muscle loss.

[0128] [Example 3] We confirmed that administration of the cell preparation of the present invention exhibits an effect of improving mitochondrial function.

[0129] The gastrocnemius muscle and soleus muscle of each mouse collected in Example 2 were examined using RT-PCR to confirm the expression levels of mRNA encoding PGC1-α, COX4, and GLUT4 in each muscle tissue using a qPCR kit (PowersSYBRR Green CR Master Mix, ThermoFisher, Cat. No.:#437659). In addition, the expression level of PGC1-α in the gastrocnemius muscle was confirmed by Western blotting. Furthermore, the expression level of PGC1-α was calculated from the stained image of the resulting gel. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an endogenous control for RT-PCR and Western blotting (the same applies hereafter). These results are shown in Figures 6 and 7. Primer set for PGC1-α Forward primer: 5'-CCGAGAATTCATGGAGCAAT-3' (SEQ ID NO: 1) Reverse primer: 5'-TTTCTGTGGGTTTGGTGTGA-3' (SEQ ID NO: 2) • Primer set for COX4 Forward primer: 5'-AGCTGAGCCAAGCAGAGAAG-3' (Sequence ID 3) Reverse primer: 5'-AATCACCAGAGCCGTGAATC-3' (SEQ ID NO: 4) • Primer set for GLUT4 Forward primer: 5'-GACGGACACTCCATCTGTTG-3' (SEQ ID NO: 5) Reverse primer: 5'-GCCACGATGGAGACATAGC-3' (SEQ ID NO: 6) GAPDH Primer Set Forward primer: 5'-ATGTGTCCGTCGTGGATCTGA-3' (SEQ ID NO: 7) Reverse primer: 5'-ATGCCTGCTTCACCACCTTCT-3' (SEQ ID NO: 8)

[0130] Figure 6 shows graphs illustrating the expression levels of PGC1-α, COX4, and GLUT4 in the gastrocnemius muscle of each mouse. In Figure 6, the top three graphs show the results in the gastrocnemius muscle, and the bottom three graphs show the results in the soleus muscle. In the top and bottom graphs of Figure 6, the results for PGC1-α, COX4, and GLUT4 are shown from left to right. In each graph of Figure 6, the vertical axis represents the relative expression level of PGC1-α, COX4, or GLUT4 mRNA compared to the endogenous control (GAPDH), and the horizontal axis represents the mouse species.

[0131] Figure 7 is a photograph showing the results of a Western blot. In Figure 7, (A) is a gel image showing the results for PGC1-α in the gastrocnemius muscle, and Figure 7(B) is a graph showing the expression level of PGC1-α in the gastrocnemius muscle. In the gel image of Figure 7(A), the upper panel shows the expression level of PGC1-α, and the lower panel shows the expression level of the endogenous control (GAPDH). Also, in the gel image of Figure 7(A), the results for Control, Example A (Cell), Reference Example (Ex), and Example B (Cell+Ex) are shown in groups of three from the left.

[0132] As shown in Figure 6, compared to control mice, the mice of Example A showed increased expression levels of PGC1-α, COX4, and GLUT4 mRNA in the gastrocnemius and soleus muscles 12 weeks after cell administration. Compared to control mice, the mice of Example B also showed increased expression levels of PGC1-α, COX4, and GLUT4 mRNA in the gastrocnemius and soleus muscles 12 weeks after cell administration.

[0133] As shown in Figures 7(A) and (B), the mice of Example A showed increased expression of PGC1-α protein in the gastrocnemius and soleus muscles 12 weeks after cell administration, compared with the control mice. Similarly, the mice of Example B showed increased expression of PGC1-α protein in the gastrocnemius muscle 12 weeks after cell administration, compared with the control and reference example mice. These results suggest that the cell preparation of the present invention suppresses muscle mass loss through its unique function of improving mitochondrial function.

[0134] Next, the morphology of mitochondria in the gastrocnemius muscle was observed using an electron microscope (JEM-1400Plus, JEOL Ltd.). The number of mitochondria per unit area was then counted. These results are shown in Figure 8.

[0135] Figure 8 shows photographs and diagrams illustrating the results of mitochondrial measurements. In Figure 8, (A) shows the results of electron microscope images of the gastrocnemius muscle, and (B) shows the results of the number of mitochondria per unit area. As shown in Figures 8(A) and (B), the mice of Examples A and B showed an increase in the number of mitochondria in the gastrocnemius muscle 12 weeks after cell administration, compared with the control and reference example mice.

[0136] Mitochondrial dysfunction has been suggested to be involved in age-related muscle loss disorders such as sarcopenia and frailty. Therefore, it is presumed that the cell preparations of the present invention will exert a therapeutic effect against age-related muscle loss disorders by improving mitochondrial function.

[0137] [Example 4] We confirmed that administration of the cell preparation of the present invention suppressed apoptosis in muscle cells.

[0138] The amount of fragmented DNA indicating cellular apoptosis in the gastrocnemius muscle and soleus muscle of each mouse collected in Example 2 was confirmed using the TUNEL (T-mediated digoxygenin (biotin)-dUTP nick end labeling) method. In addition, the expression levels of cleaved-caspase-3 and cleaved-caspase-8, markers of endogenous apoptosis, were confirmed by Western blotting. These results are shown in Figure 9.

[0139] Figure 9 shows photographs and graphs illustrating the results of apoptosis analysis. In Figure 9, (A) shows photographs illustrating the results of TUNEL staining, with the four figures on the left showing the results for the gastrocnemius muscle and the four figures on the right showing the results for the soleus muscle. In the four figures on the left and right of Figure 9(A), the upper left shows the results for the control, the upper right shows the results for Example A (Cell), the lower left shows the results for Reference Example (Ex), and the lower right shows the results for Example B (Cell + Ex). In Figure 9(A), fragmented DNA stained by TUNEL staining is indicated by a white triangle (▽). Figure 9(B) is a gel image and graph showing the expression level of Cleaved-caspase-3 in the gastrocnemius muscle. Figure 9(C) is a gel image and graph showing the expression level of Cleaved-caspase-8 in the gastrocnemius muscle. In the graphs in Figures 9(B) and (C), the vertical axis shows the relative expression levels of Cleaved-caspase-3 or Cleaved-caspase-8 compared to the endogenous control (GAPDH), and the horizontal axis shows the mouse species. As shown in Figure 9, compared to the control mouse, the mice of Examples A and B showed a decrease in fragmented DNA, which indicates apoptosis in each muscle tissue, at 12 weeks after cell administration. Furthermore, compared to the control and reference example mice, the mice of Examples A and B showed decreased expression levels of Cleaved-caspase-3 and Cleaved-caspase-8, which are markers of endogenous apoptosis, in each muscle tissue at 12 weeks after cell administration.

[0140] It is known that patients with sarcopenia have increased apoptosis of muscle cells. The cell preparation of the present invention can suppress apoptosis of muscle cells, for example, and thus suppress the decrease in muscle mass, thereby exhibiting a therapeutic effect on sarcopenia. Furthermore, since muscle mass decreases due to apoptosis of muscle cells in age-related muscle loss disorders such as frailty, it can be said that the cell preparation of the present invention will also exhibit a therapeutic effect in other age-related muscle loss disorders.

[0141] [Example 5] We confirmed that the cell preparation of the present invention exhibits anti-inflammatory effects upon administration.

[0142] In the gastrocnemius muscle and soleus muscle tissues of each mouse collected in Example 2, the mRNA expression levels of the inflammatory cytokine Tumor necrosis factor-α (TNF-α) and the chemokine Monocyte chemotactic protein-1 (MCP-1) were confirmed by RT-PCR using the qPCR kit (PowersSYBRR Green CR Master Mix, ThermoFisher, Cat. No.: #437659). Furthermore, CD68 staining was performed on the collected muscle tissues to detect macrophages. These results are shown in Figure 10. TNF-α Primer Set Forward primer: 5'-GACTTTCTCCTGGTATGAGATAG-3' (SEQ ID NO: 9) Reverse primer: 5'-AGGCTGCCCCGACTACGT-3' (SEQ ID NO: 10) Primer set for MCP-1 Forward primer: 5'-GCCCCACTCACCTGCTGCTACT-3' (SEQ ID NO: 11) Reverse primer: 5'-CCTGCTGCTGGTGATCCTCTTGT-3' (SEQ ID NO: 12)

[0143] Figure 10 shows graphs and photographs of muscle tissue illustrating the expression levels of inflammatory cytokines. Figure 10(A) shows graphs illustrating the expression levels of TNF-α or MCP-1 in the gastrocnemius and soleus muscles of each mouse. In Figure 10(A), the top two graphs show the results for the gastrocnemius muscle, and the bottom two graphs show the results for the soleus muscle. In the top and bottom graphs of Figure 10(A), the results for TNF-α and MCP-1 are shown from left to right. In each graph of Figure 10(A), the vertical axis represents the relative expression level of TNF-α or MCP-1 mRNA compared to the endogenous control (GAPDH), and the horizontal axis represents the mouse species. Figure 10(B) shows photographs illustrating the results of CD68 staining, with the four left graphs showing the results for the gastrocnemius muscle and the four right graphs showing the results for the soleus muscle. In the four figures on the left and right of Figure 10(B), the upper left shows the results for the control, the upper right shows the results for Example A (Cell), the lower left shows the results for the reference example (Ex), and the lower right shows the results for Example B (Cell + Ex).

[0144] As shown in Figure 10, the mice of Examples A and B showed reduced expression levels of TNF-α or MCP-1 mRNA in the gastrocnemius and soleus muscles 12 weeks after cell administration, compared to the control mice. Furthermore, CD68 staining revealed that the mice of Examples A and B had a reduced amount of macrophages in the gastrocnemius and soleus muscles compared to the control.

[0145] In the mice of Examples A and B, the expression levels of TNF-α and MCP-1 in muscle tissue decreased, and the amount of macrophages in muscle tissue decreased, indicating that the cell preparation of the present invention can suppress inflammation in muscle tissue. It is said that as aging progresses, micro-inflammation occurs in muscle tissue, which leads to a decrease in muscle mass. Therefore, it was found that the cell preparation of the present invention can suppress the decrease in muscle mass by suppressing inflammation in muscle tissue, thereby exhibiting a therapeutic effect on sarcopenia.

[0146] [Example 6] We confirmed that administration of the cell preparation of the present invention improves the proliferation of muscle cells.

[0147] The gastrocnemius muscle and soleus muscle of each mouse collected in Example 2 were examined using the qPCR kit (PowersSYBRR Green CR Master Mix, ThermoFisher, Cat. No.: #437659) to confirm the expression level of TGF-β1 mRNA in the muscle tissue by RT-PCR. In addition, PCNA (proliferating cell nuclear antigen) staining was performed on each collected muscle tissue to detect the proliferating cell nuclear antigen in each muscle tissue, and the expression of desmin and laminin 5 in each muscle tissue was confirmed by Desmin+ / Laminin5+ double fluorescence staining. These results are shown in Figure 11. TGF-β1 primer set Forward primer: 5'-TGGAGCAACATGTGGAACTC-3' (SEQ ID NO: 13) Reverse primer: 5'-GTCAGCAGCCGGTTACCA-3' (SEQ ID NO: 14)

[0148] Figure 11 shows graphs and photographs of muscle tissue illustrating TGF-β1 expression levels. In Figure 11, (A) is a graph showing TGF-β1 expression levels in the gastrocnemius and soleus muscles of each mouse, and (B) is a photograph showing the results of PCNA staining. In Figure 11(A), the graph on the left shows the results in the gastrocnemius muscle, and the graph on the right shows the results in the soleus muscle. In each figure of Figure 11(A), the vertical axis shows the relative expression level of TGF-β1 mRNA compared to the endogenous control (GAPDH), and the horizontal axis shows the mouse species. In Figure 11(B), the four figures on the left show the results for the gastrocnemius muscle, and the four figures on the right show the results for the soleus muscle. In the four figures on the left and right sides of Figure 11(B), the upper left shows the results for the control, the upper right shows the results for Example A (Cell), the lower left shows the results for the reference example (Ex), and the lower right shows the results for Example B (Cell + Ex). In Figure 11(B), areas of proliferating cell nuclear antigen stained by PCNA staining are indicated by white triangles (▽). Figure 11(C) is a photograph showing the results of double fluorescence staining of Desmin / Laminin5, with the four figures on the left showing the results for the gastrocnemius muscle and the four figures on the right showing the results for the soleus muscle. In the four figures on the left and right sides of Figure 11(C), the upper left shows the results for the control, the upper right shows the results for Example A (Cell), the lower left shows the results for the reference example (Ex), and the lower right shows the results for Example B (Cell + Ex). In Figure 11(C), Desmin stained by double fluorescence staining of Desmin / Laminin5 + / Laminin5 + The parts marked (double positive) are indicated with a white asterisk (*).

[0149] As shown in Figure 11, compared to the control mouse, the mice of Examples A and B showed increased TGF-β1 mRNA expression in the gastrocnemius and soleus muscles 12 weeks after cell administration. Furthermore, PCNA staining revealed that, compared to the control mouse, the number of proliferating cell nuclear antigens in the gastrocnemius and soleus muscles increased 12 weeks after cell administration, indicating enhanced skeletal muscle cell proliferation. In addition, Desmin+ / Laminin5+ double fluorescence staining revealed that, compared to the control mouse and the reference mouse, the mice of Examples A and B showed increased expression of Desmin and Laminin5 in the gastrocnemius and soleus muscles 12 weeks after cell administration, indicating induced skeletal muscle repair.

[0150] These results indicate that the cell preparation of the present invention induces the proliferation of skeletal muscle cells, thereby promoting the repair of skeletal muscle. Furthermore, since TGF-β1 induces the proliferation of skeletal muscle cells, it was presumed that the cell preparation of the present invention induces the above pathway through the induction of TGF-β1 expression. In patients with age-related muscle loss such as sarcopenia and frailty, it is known that the number of skeletal muscle cells decreases. Therefore, it was found that the cell preparation of the present invention induces the proliferation of skeletal muscle cells, thereby reducing the decrease in muscle mass and thus providing a therapeutic effect on age-related muscle loss.

[0151] [Example 7] We confirmed that administration of the cell preparation of the present invention enhanced skeletal muscle repair.

[0152] The gastrocnemius muscle and soleus muscle of each mouse collected in Example 2 were examined using the qPCR kit (PowersSYBRR Green CR Master Mix, ThermoFisher, Cat. No.: #437659) to confirm the expression levels of mRNA encoding cathepsin K (CatK) and GAPDH using RT-PCR. Furthermore, the expression levels of Sirt1 and myosin heavy chain (MHC), as well as GAPDH, were confirmed by Western blotting. These results are shown in Figure 12. • Primer set for CatK Forward primer: 5'-AGCAGGCTGGAGGACTAAGGT-3' (SEQ ID NO: 15) Reverse primer: 5'-TTTGTGCATCTCAGTGGAAGACT-3' (SEQ ID NO: 16)

[0153] Figure 12 shows graphs or photographs illustrating the expression levels of CatK, Sirt1, and MHC. In Figure 12, (A) is a graph showing the expression levels of CatK in the gastrocnemius and soleus muscles of each mouse, (B) is a gel image and graph showing the expression level of Sirt1 in the gastrocnemius muscle, and (C) is a gel image and graph showing the expression level of MHC in the gastrocnemius muscle. In Figure 12(A), the left graph shows the results in the gastrocnemius muscle, and the right graph shows the results in the soleus muscle. In each figure of Figure 12(A), the vertical axis shows the relative expression level of CatK to the endogenous control (GAPDH), and the horizontal axis shows the mouse species. In the gel images of Figures 12(B) and (C), the upper panel shows the expression level of Sirt1 or MHC, and the lower panel shows the expression level of the endogenous control (GAPDH). Furthermore, in each gel image, the results for Control, Example A (Cell), Reference Example (Ex), and Example B (Cell+Ex) are shown in three lanes from left to right, respectively. In the graphs of Figure 12(B) and (C), the vertical axis shows the relative expression level of Sirt1 or MHC compared to the endogenous control (GAPDH), and the horizontal axis shows the mouse species.

[0154] As shown in Figure 12(A), the mice of Examples A and B showed a significant decrease in CatK expression in each muscle tissue at 12 weeks after cell administration, compared with the control mice. Furthermore, the mice of Examples A and B showed a significant increase in Sirt1 and MHC expression at 12 weeks after cell administration, compared with the control and reference example mice.

[0155] CatK is known to be highly expressed after muscle injury, inducing inflammation, thereby exacerbating muscle damage, and delaying regeneration. Sirtuin genes such as Sirt1 are known to be associated with anti-aging effects. MHC is a gene associated with muscle mass repair and increase, and is known to have a function in maintaining the amount of muscle fibers. Therefore, it was hypothesized that the cell preparation of the present invention can suppress the expression of CatK in skeletal muscle and increase the expression of Sirt1 and MHC, thereby enhancing and maintaining skeletal muscle repair. Thus, it was found that the cell preparation of the present invention suppresses the expression of genes that exacerbate muscle damage, while inducing the expression of genes that regenerate muscle, thereby suppressing the decrease in muscle mass and thus exhibiting a therapeutic effect on sarcopenia.

[0156] [Example 8] We confirmed that the exosomes in the cell preparation of the present invention can suppress apoptosis in muscle cells.

[0157] (1) Purification of exosomes from cell preparation culture supernatant To confirm that the exosomes in the cell preparation of the present invention suppress apoptosis, exosomes were first purified from the culture supernatant of the cell preparation. Specifically, cell preparations prepared in the same manner as in Examples 1(1) to (3) were placed in a Corning® CellBIND® surface 100 mm dish (Corning, Product Number: #3292) with 1 × 10 cells. 5The cells were seeded at a rate of cells / 1ml and cultured for 48 hours. The culture medium used was CiMS(trademark)-BM (Nipro Corporation, product code: 87-070) with CiMS(trademark)-sAF (Nipro Corporation, product code: 87-072), an animal-free additive for human mesenchymal stem cell basal culture medium, added in the proportion specified in the package insert. After the culture, a first centrifugation was performed at 300×g for 10 minutes to remove the cells and obtain the first supernatant. The supernatant was then centrifuged a second time at 2000×g for 10 minutes to remove cell fragments and other debris and obtain the second supernatant. Subsequently, the second supernatant was filtered using a 0.22 μm filter to obtain the sample. The sample was placed in an ultracentrifuge tube (UC tube), mounted on a rotor (SW32Ti-12U, Beckman Coulter), and a third centrifuge was performed using an ultracentrifuge (Optima L-100, Beckman Coulter). The third centrifuge was performed at 35,000 rpm for 70 minutes. After the third centrifuge, the supernatant was removed to obtain a precipitate. 1 ml of filtered PBS was added to the precipitate and vortexing was performed. After vortexing, the volume was made up to 35 ml with PBS, and a fourth centrifuge was performed under the same conditions as the third centrifuge. After the fourth centrifuge, the supernatant was removed, a small amount of PBS was added, and vortexing was performed to obtain an exosome solution. The exosome solution was then stored in a 1.5 ml low-adsorption tube.

[0158] (2) Electron microscopy of exosomes Next, to confirm whether the exosome solution contained exosomes, the exosome solution was observed using an electron microscope. Specifically, 10 μl of the exosome solution was placed on a grid and air-dried for 10 minutes. After air-drying, uranyl acetate was added and negative staining was performed. After negative staining, observation was performed using a transmission electron microscope (JEM-1400PLUS, JEOL Ltd.). These results are shown in Figure 13.

[0159] Figure 13 is an electron microscope image of exosomes. In Figure 13, the scale bar represents 200 nm. As shown in Figure 13, numerous round vesicles with diameters ranging from approximately 50 mm to 200 mm were observed. From the above, it is suggested that exosomes can be purified from the culture supernatant of the cell preparation of the present invention.

[0160] (3) Confirmation of exosome markers To confirm whether the round vesicles observed with the electron microscope were exosomes, Western blotting was used to check whether the vesicles expressed exosome markers. Specifically, 50 μl of 2× sample buffer was added to 50 μl of the exosome solution obtained in Example 8(1), and the mixture was heated at 95°C for 5 minutes to prepare the sample. After preparation, the sample was applied to the wells of a 10-20% SuperSep™ Ace (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) acrylamide gel, and electrophoresis was performed. After electrophoresis, Western blotting was performed. For detection of exosomes in Western blotting, the primary antibody was reacted under overnight conditions, and then stained with a secondary antibody at room temperature (approximately 25°C) for 1 hour. The primary antibodies used were anti-CD9 monoclonal antibody (clone: ​​1K, 1000-fold dilution, Cat. No: 041-27763, Fujifilm Wako Pure Chemical Industries, Ltd.), anti-CD63 monoclonal antibody (clone: ​​3-13, 1000-fold dilution, Cat. No: 012-27063, Fujifilm Wako Pure Chemical Industries, Ltd.), and anti-CD81 monoclonal antibody (clone: ​​17B1, 1000-fold dilution, Cat. No: 011-27773, Fujifilm Wako Pure Chemical Industries, Ltd.). The secondary antibody used was Anti-Mouse IgG, HRP-Linked F(ab')2 Fragment Sheep (5000-fold dilution, Cat. No: NA9310V, Cytiva). Similarly, exosome markers were detected in three additionally prepared exosome solutions. These results are shown in Figure 14.

[0161] Figure 14 is a photograph showing the detection results of exosome markers by Western blotting. In Figure 14, the left side of the photograph shows the exosome markers, the top of the photograph shows the exosome solution samples, and lanes 1 to 4 show the results of independently prepared exosome solutions. As shown in Figure 14, exosome markers were detected in all of the exosome solution samples, and the secretion of exosomes by umbilical cord-derived mesenchymal stem cells was reliably confirmed. From the above, it was found that exosomes can be purified from the culture supernatant of the cell preparation of the present invention.

[0162] (4) Evaluation of the particle size distribution of exosomes The particle size distribution of exosomes obtained from the culture supernatant of the cell preparation of the present invention was evaluated. Specifically, the exosome solution obtained in Example 8(1) was diluted 50-fold with PBS. After the dilution, the particle size of the exosomes was measured using a particle size analyzer (Nano particle tracking analysis (NanoSight), Malvern Panalytical) with default parameters. These results are shown in Figure 15.

[0163] Figure 15 is a graph showing the particle size distribution of the exosome marker. In Figure 15, the vertical axis represents the exosome concentration (number of vesicles / ml), and the horizontal axis represents the exosome size (nm). As shown in Figure 15, the exosomes of the umbilical cord-derived mesenchymal cells were distributed in the range of 40 to 400 nm, with the most abundant being 116 nm, the smallest being 40 nm, and the largest being 621 nm. In addition, many exosomes were observed in the range of approximately 100 nm to approximately 300 nm. From the above, it was found that the exosomes obtained from the culture supernatant of the cell preparation of the present invention are exosomes in the range of approximately 100 nm to approximately 300 nm.

[0164] (5) Evaluation of exosome uptake into cultured muscle cells We evaluated whether exosomes obtained from the culture supernatant of the cell preparation of the present invention could be taken up by cultured muscle cells. Specifically, the exosome solution (10 μg of protein) obtained in Example 8(1) was labeled using the ExoSparkler Exosome Membrane Labeling Kit-Green (Cat. No: EX01, manufactured by Dojin Chemical Laboratories). After labeling, 1.25 × 10⁶ exosomes were placed in a dish. 4 The solution was added to seeded C2C12 cells (mouse striated muscle cells). After the addition, the cells were incubated for 24 hours. After incubation, the cells were fixed using 4% paraformaldehyde-containing phosphate buffer (Cat. No: 161-20141, Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature for 20 minutes. After fixation, the cells were washed with PBS at room temperature for 5 minutes, and this washing was repeated a total of three times. Subsequently, the cells were observed using a fluorescence microscope (Super-resolution / confocal Microscopy (LSM880-ELYRA PS. 1, Carl Zeiss)). These results are shown in Figure 16.

[0165] Figure 16 is a photograph showing C2C12 cells that have taken up exosomes. As shown in Figure 16, it was found that the C2C12 cells took up labeled exosomes. From the above, it was found that exosomes obtained from the culture supernatant of the cell preparation of the present invention are taken up by muscle cells.

[0166] (6) Evaluation of apoptosis suppression by exosomes We investigated whether exosomes obtained from the culture supernatant of the cell preparation of the present invention suppress apoptosis in cultured muscle cells that have taken up the exosomes. Specifically, we placed 2 × 10⁶ C2C12 cells (mouse striated muscle cells) in a 12-well culture plate that had a coverslip. 4After seeding to form cells, 300 μmol / l H2O2 was added. Following the addition, incubation was performed for 12 hours. After incubation, the cells were cultured in DMEM medium to which the exosome solution (1 μg of protein) obtained in Example 8(1) had been added. For the negative control, instead of the exosome solution, an equal volume of PBS was added to DMEM medium for cultivation. This cultivation was performed for 12 hours. After cultivation, TUNEL staining was performed, and the cells were observed using a fluorescence microscope. ProLong was used for nuclear staining. TM Galss Antifade Mountan with NucBlue TM A ThermoFisher (Cat.No:P36985) was used. The proportion of TUNEL-positive cells among nuclear-positive cells was calculated. These results are shown in Figure 17. t-tests (and nonparametric tests) were used to test for statistical significance.

[0167] Figure 17 shows photographs and graphs illustrating apoptosis in C2C12 cells that have taken up exosomes. In Figure 17, (A) is a photograph showing TUNEL staining with and without exosome addition, and (B) is a graph showing the percentage of TUNEL-positive cells with and without exosome addition. In Figure 17(A), the left side of the photograph shows whether or not exosomes were added, and the top of the photograph shows the type of marker. In Figure 17(B), the vertical axis shows the percentage of TUNEL-positive cells (%), and the horizontal axis shows whether or not exosomes were added. As shown in Figure 17(A), fewer TUNEL-positive cells were observed in C2C12 cells with exosome addition (Exosome) compared with negative controls (Control) without exosome addition. Also, as shown in Figure 17(B), the percentage of TUNEL-positive cells was significantly reduced in C2C12 cells with exosome addition compared with negative controls without exosome addition. From the above, it was found that exosomes obtained from the culture supernatant of the cell preparation of the present invention can suppress apoptosis caused by H2O2 in muscle cells that have taken up the exosomes. Furthermore, the inventors have shown for the first time that umbilical cord-derived cells secrete exosomes.

[0168] Although the present invention has been described above with reference to embodiments and examples, the present invention is not limited to the above embodiments and examples. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention.

[0169] This application claims priority based on Japanese Patent Application No. 2020-190551, filed on 16 November 2020, and incorporates all of its disclosures herein.

[0170] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. (Note 1) A cell preparation used to suppress the decrease in muscle mass, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 2) The umbilical cord-derived cells are umbilical cord-derived mesenchymal cells, as described in Appendix 1 of the cell preparation. (Note 3) The umbilical cord-derived cells are, (i) Positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) Cell preparations as described in Appendix 1 or 2, which are negative for CD45, CD34, CD11b, CD19, and HLA-Class II. (Note 4) The umbilical cord-derived cells are, (iii) A cell preparation according to any one of the appendices 1 to 3, wherein the expression of one of the IDO, PGE2, or PD-L1 gene and / or protein is induced under inflammatory conditions. (Note 5) The cell preparation according to any one of the appendices 1 to 4, wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue including the amnion, blood vessels, perivascular tissue and / or Wharton's jelly. (Note 6) The cell preparation is 1 × 10 6 ~1 × 10 9 A cell preparation according to any one of the appendices 1 to 5, containing umbilical cord-derived cells. (Note 7) The cell preparation described above is the cell preparation described in any of Appendix 1 to 6, comprising an extract and / or secretion of the umbilical cord-derived cells. (Note 8) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 7, which exhibits an effect of improving mitochondrial function in muscle tissue. (Note 9) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 8, which exhibits an effect of increasing the number of mitochondria in muscle tissue. (Note 10) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 9, which exhibits an effect of inducing the expression of genes that improve mitochondrial function in muscle tissue. (Note 11) The mitochondrial function-enhancing genes are the PGC1-α gene, the COX4 gene, and / or the GLUT4 gene, according to the cell preparation described in Appendix 10. (Note 12) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 11, which exhibits an inhibitory effect on apoptosis of muscle cells. (Note 13) The cell preparation is a cell preparation according to any one of the appendices 1 to 12, which exhibits anti-inflammatory activity in muscle tissue. (Note 14) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 13, which exhibits an inhibitory effect on the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue. (Note 15) The inflammatory cytokine gene is the TNF-α gene, as described in Appendix 14 of the cell preparation. (Note 16) The cell preparation described in Appendix 14 is the MCP-1 gene, where the chemokine gene is the MCP-1 gene. (Note 17) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 16, which exhibits a proliferation-promoting effect on muscle cells. (Note 18) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 17, which exhibits an effect of inducing the expression of gene that promotes the proliferation of muscle cells. (Note 19) The cell preparation described in Appendix 18 has the TGF-β1 gene as the gene that promotes the proliferation of muscle cells. (Note 20) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 19, which exhibits a muscle tissue repair effect. (Note 21) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 20, which exhibits an effect of inducing the expression of muscle tissue repair genes. (Note 22) The cell preparation described in Appendix 21, wherein the muscle tissue repair genes are the SIRT1 gene and / or MHC genes. (Note 23) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 22, which exhibits an inhibitory effect on muscle tissue damage. (Note 24) The cell preparation described above is a cell preparation according to any one of the appendices 1 to 23, which exhibits an inhibitory effect on the expression of gene that induces muscle tissue damage. (Note 25) The gene that induces muscle tissue damage is the cathepsin K gene, as described in the cell preparation described in Appendix 24. (Note 26) A cell preparation described in any of Appendix 1 to 25, to be used in conjunction with exercise therapy. (Note 27) A cell preparation described in any of Appendix 1 to 26, for use in subjects with inflammation in muscle tissue. (Note 28) A cell preparation described in any of the appendices 1 to 27, for intravenous administration. (Note 29) A cell preparation as described in any of Appendix 1 to 28, wherein the decrease in muscle mass is due to aging. (Note 30) A cell preparation described in any of Appendix 1 to 29, for administration to middle-aged and elderly individuals. (Note 31) A cell preparation used in the treatment of age-related muscle loss, A cell preparation comprising a cell preparation used to suppress the decrease in muscle mass as described in any of the appendices 1 to 30. (Note 32) The cell preparation described in Appendix 31, wherein the age-related muscle loss is sarcopenia or frailty. (Note 33) A cell preparation used to improve mitochondrial function in muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 34) A cell preparation used to increase the number of mitochondria in muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 35) A cell preparation used to induce the expression of genes that improve mitochondrial function in muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 36) The mitochondrial function-enhancing genes are the PGC1-α gene, the COX4 gene, and / or the GLUT4 gene, as described in Appendix 35 of the cell preparation. (Note 37) A cell preparation used to suppress apoptosis in muscle cells, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 38) A cell preparation used for anti-inflammatory (inflammation suppression) purposes in muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 39) A cell preparation used to suppress the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 40) The inflammatory cytokine gene is the TNF-α gene, as described in Appendix 39 of the cell preparation. (Note 41) The cell preparation described in Appendix 39 is the MCP-1 gene, where the chemokine gene is the MCP-1 gene. (Note 42) A cell preparation used to promote the proliferation of muscle cells, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 43) A cell preparation used to induce the expression of gene that promotes muscle cell proliferation, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 44) The cell preparation described in Appendix 43 has the TGF-β1 gene as the gene that promotes the proliferation of muscle cells. (Note 45) A cell preparation used for muscle tissue repair, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 46) A cell preparation used to induce the expression of muscle tissue repair genes, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 47) The cell preparation described in Appendix 46, wherein the muscle tissue repair genes are the SIRT1 gene and / or MHC genes. (Note 48) A cell preparation used to suppress muscle tissue damage, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 49) A cell preparation used to suppress the expression of gene that induces muscle tissue damage, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 50) The gene that induces muscle tissue damage is the cathepsin K gene, as described in Appendix 49 of the cell preparation. (Note 51) A cell preparation used to suppress interstitial fibrosis in target muscle tissue, The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 52) A method for suppressing muscle mass loss in a subject, A method that uses a cell preparation used to suppress the decrease in muscle mass, as described in any of the appendices 1 to 30. (Note 53) The method according to Appendix 52, wherein the subject is administered a cell preparation used to suppress the decrease in muscle mass as described in any of Appendix 1 to 30. (Note 54) A treatment method for individuals experiencing a decrease in muscle mass, A method that uses a cell preparation used to suppress the decrease in muscle mass, as described in any of the appendices 1 to 30. (Note 55) The method according to Appendix 54, wherein the subject is administered a cell preparation used to suppress the decrease in muscle mass as described in any of Appendix 1 to 30. (Note 56) A method for improving mitochondrial function in target muscle tissue, A method that uses cell preparations used to improve mitochondrial function in muscle tissue, as described in Appendix 33. (Note 57) The method according to Appendix 56, wherein the subject is administered the cell preparation described in Appendix 33. (Note 58) A method for increasing the number of mitochondria in target muscle tissue, A method that uses a cell preparation used to increase the number of mitochondria in muscle tissue as described in Appendix 34. (Note 59) The method described in Appendix 58, wherein the subject is administered the cell preparation described in Appendix 34. (Note 60) A method for inducing the expression of genes that improve mitochondrial function in target muscle tissue, A method that uses a cell preparation used to induce the expression of mitochondrial function-enhancing genes in muscle tissue, as described in Appendix 35 or 36. (Note 61) The method according to Appendix 60, wherein the subject is administered the cell preparation described in Appendix 35 or 36. (Note 62) A method for suppressing apoptosis in target muscle cells, A method that uses a cell preparation used to suppress apoptosis in muscle cells as described in Appendix 37. (Note 63) The method according to Appendix 62, wherein the subject is administered the cell preparation described in Appendix 37. (Note 64) A method for suppressing inflammation in target muscle tissue, A method that uses cell preparations used for anti-inflammatory (inflammation suppression) effects in muscle tissue as described in Appendix 38. (Note 65) The method described in Appendix 64, wherein the subject is administered the cell preparation described in Appendix 38. (Note 66) A method for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in target muscle tissue, A method of using a cell preparation for suppressing the expression of an inflammatory cytokine gene and / or a chemokine gene in the muscle tissue described in Supplementary Note 39 in a subject. (Supplementary Note 67) The method described in Supplementary Note 66 of administering the cell preparation described in Supplementary Note 39 to a subject. (Supplementary Note 68) A method for promoting the proliferation of muscle cells in a subject, A method of using a cell preparation for promoting the proliferation of muscle cells described in Supplementary Note 42 in a subject. (Supplementary Note 69) The method described in Supplementary Note 68 of administering the cell preparation described in Supplementary Note 42 to a subject. (Supplementary Note 70) A method for inducing the expression of a gene for promoting the proliferation of muscle cells in a subject, A method of using a cell preparation for inducing the expression of a gene for promoting the proliferation of muscle cells described in Supplementary Note 43 or 44 in a subject. (Supplementary Note 71) The method described in Supplementary Note 70 of administering the cell preparation described in Supplementary Note 43 or 44 to a subject. (Supplementary Note 72) A method for repairing muscle tissue in a subject, A method of using a cell preparation for repairing muscle tissue described in Supplementary Note 45 in a subject. (Supplementary Note 73) The method described in Supplementary Note 72 of administering the cell preparation described in Supplementary Note 45 to a subject. (Supplementary Note 74) A method for inducing the expression of a gene for repairing muscle tissue in a subject, A method of using a cell preparation for inducing the expression of a gene for repairing muscle tissue described in Supplementary Note 46 or 47 in a subject. (Supplementary Note 75) The method described in Supplementary Note 74 of administering the cell preparation described in Supplementary Note 46 or 47 to a subject. (Supplementary Note 76) A method for suppressing damage to muscle tissue in a subject, A method of using a cell preparation for suppressing damage to muscle tissue described in Supplementary Note 48 in a subject. (Supplementary Note 77) The method according to Appendix 76, wherein the subject is administered the cell preparation described in Appendix 48. (Note 78) A method for suppressing the expression of a gene that induces damage in the target muscle tissue, A method that uses a cell preparation used to suppress the expression of muscle tissue damage-inducing genes as described in Appendix 49 or 50. (Note 79) The method according to Appendix 78, wherein the subject is administered a cell preparation described in Appendix 49 or 50. (Note 80) A method for treating patients with age-related muscle loss, A treatment method comprising administering a cell preparation described in any of Appendix 1 to 30 to the aforementioned patient with age-related muscle loss. (Note 81) A method for suppressing interstitial fibrosis of target muscle tissue, A method that uses a cell preparation used to suppress interstitial fibrosis in the target muscle tissue as described in Appendix 51. (Note 82) The method according to Appendix 81, wherein the subject is administered the cell preparation described in Appendix 51. (Note 83) This is a cell composition used to suppress the decrease in muscle mass. The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 84) This is a cell preparation used for the treatment of age-related muscle loss. The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 85) This is a cell preparation used to improve mitochondrial function in muscle tissue. The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 86) This is a cell preparation used to increase the number of mitochondria in muscle tissue. The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 87) This is a cell preparation used to induce the expression of genes that improve mitochondrial function in muscle tissue. The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 88) A cell preparation for use in suppressing apoptosis in muscle cells, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 89) A cell preparation for use in suppressing inflammation in muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 90) A cell preparation for use in suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 91) A cell preparation for use in promoting the proliferation of muscle cells, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 92) A cell preparation for use in inducing the expression of genes promoting the proliferation of muscle cells, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 93) A cell preparation for use in repairing muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 94) A cell preparation for use in inducing the expression of genes for repairing muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 95) A cell preparation for use in suppressing damage to muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 96) A cell preparation for use in suppressing the expression of genes inducing damage to muscle tissue, The cell preparation is a cell preparation containing cells derived from umbilical cord. (Appendix 97) This is a cell preparation used to suppress fibrosis of muscle tissue interstitial tissue. The cell preparation is a cell preparation containing umbilical cord-derived cells. (Note 98) A pharmaceutical composition comprising exosomes derived from umbilical cord cells and a pharmaceutically acceptable carrier. (Note 99) The pharmaceutical composition according to Appendix 98, wherein the average diameter of the exosomes is 1 to 500 nm. (Note 100) The exosome comprises a lipid membrane and a lumen. The lipid membrane is derived from the umbilical cord-derived cells, The pharmaceutical composition according to Appendix 98 or 99, wherein the lumen contains the cytoplasm of the umbilical cord-derived cells. (Note 101) A pharmaceutical composition described in any of appendices 98 to 100, for use in suppressing the decrease in muscle mass. (Note 102) A pharmaceutical composition according to any one of the appendices 98 to 101, for use in improving mitochondrial function in muscle tissue. (Note 103) A pharmaceutical composition according to any one of the appendices 98 to 102, for use in increasing the number of mitochondria in muscle tissue. (Note 104) A pharmaceutical composition according to any one of Appendix 98 to 103, for use in inducing the expression of genes that improve mitochondrial function in muscle tissue. (Note 105) A pharmaceutical composition according to any one of the appendices 98 to 104, for use in suppressing apoptosis of muscle cells. (Note 106) A pharmaceutical composition described in any of Appendix 98 to 105 for use in anti-inflammatory (inhibitory) purposes in muscle tissue. (Note 107) A pharmaceutical composition according to any one of the appendices 98 to 106, for use in suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue. (Note 108) A pharmaceutical composition described in any of appendices 98 to 107, for use in promoting the proliferation of muscle cells. (Note 109) A pharmaceutical composition according to any one of the appendices 98 to 108, for use in inducing the expression of gene that promotes the proliferation of muscle cells. (Note 110) A pharmaceutical composition described in any of the appendices 98 to 109, for use in the repair of muscle tissue. (Note 111) A pharmaceutical composition according to any one of the appendices 98 to 110, for use in inducing the expression of muscle tissue repair genes. (Note 112) A pharmaceutical composition according to any one of the appendices 98 to 111, for use in suppressing damage to muscle tissue. (Note 113) A pharmaceutical composition according to any one of the appendices 98 to 112, for use in age-related muscle loss. [Industrial applicability]

[0171] As described above, the present invention can suppress the decrease in muscle mass. Therefore, the present invention can treat diseases caused by a decrease in muscle mass. For this reason, the present invention is extremely useful, for example, in the pharmaceutical field.

Claims

1. A cell preparation for use in the prevention or treatment of sarcopenia or frailty, The cell preparation comprises umbilical cord-derived cells grown in a culture medium containing a serum substitute. The umbilical cord-derived cells are, (i) Positive for CD105, CD73, CD90, CD44, HLA-Class I and PD-L2, and (ii) CD45, CD34, CD11b, CD19 and HLA-Class II are negative. The umbilical cord-derived cells have an effect of inducing the expression of the SIRT1 gene and / or MHC gene in muscle tissue. Cell preparation.

2. The cell preparation according to claim 1, wherein the umbilical cord-derived cells are umbilical cord-derived mesenchymal cells.

3. The umbilical cord-derived cells are (i) Positive for CD105, CD73, CD90, CD44, HLA-Class I, HLA-G5 and PD-L2, and (ii) The cell preparation according to claim 1 or 2, wherein the cell preparation is negative for CD45, CD34, CD11b, CD19, and HLA-Class II.

4. The umbilical cord-derived cells are (iii) The cell preparation according to any one of claims 1 to 3, wherein under inflammatory conditions, the expression of at least one gene and / or protein selected from IDO and PD-L1 is induced, and / or the secretion of PGE2 is induced.

5. The cell preparation according to any one of claims 1 to 4, wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue including the amnion, blood vessels, perivascular tissue and / or Wharton's jelly.

6. The cell preparation according to any one of claims 1 to 5, wherein the cell preparation comprises 1 × 10⁶ to 1 × 10⁹ umbilical cord-derived cells.

7. The cell preparation according to any one of claims 1 to 6, comprising an extract and / or secretion of the umbilical cord-derived cells.

8. The cell preparation according to any one of claims 1 to 7, wherein the cell preparation exhibits an effect of improving mitochondrial function in muscle tissue.

9. The cell preparation according to any one of claims 1 to 8, wherein the cell preparation exhibits an effect of increasing the number of mitochondria in muscle tissue.

10. The cell preparation according to any one of claims 1 to 9, wherein the cell preparation exhibits an effect of inducing the expression of genes that improve mitochondrial function in muscle tissue.

11. The cell preparation according to claim 10, wherein the mitochondrial function-enhancing gene is the PGC1-α gene, the COX4 gene, and / or the GLUT4 gene.

12. The cell preparation according to any one of claims 1 to 11, wherein the cell preparation exhibits an inhibitory effect on apoptosis of muscle cells.

13. The cell preparation according to any one of claims 1 to 12, wherein the cell preparation exhibits an anti-inflammatory effect in muscle tissue.

14. The cell preparation according to any one of claims 1 to 13, wherein the cell preparation exhibits an inhibitory effect on the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue.

15. The cell preparation according to claim 14, wherein the inflammatory cytokine gene is the TNF-α gene.

16. The cell preparation according to claim 14, wherein the chemokine gene is the MCP-1 gene.

17. The cell preparation according to any one of claims 1 to 16, wherein the cell preparation exhibits a proliferation-promoting effect on muscle cells.

18. The cell preparation according to any one of claims 1 to 17, wherein the cell preparation exhibits an effect of inducing the expression of gene that promotes the proliferation of muscle cells.

19. The cell preparation according to claim 18, wherein the gene that promotes the proliferation of muscle cells is the TGF-β1 gene.

20. The cell preparation according to any one of claims 1 to 19, wherein the cell preparation exhibits a muscle tissue repair effect.

21. The cell preparation according to any one of claims 1 to 20, wherein the cell preparation exhibits an inhibitory effect on muscle tissue damage.

22. The cell preparation according to any one of claims 1 to 21, wherein the cell preparation exhibits an inhibitory effect on the expression of gene that induces muscle tissue damage.

23. The cell preparation according to claim 22, wherein the gene that induces muscle tissue damage is the cathepsin K gene.

24. A cell preparation according to any one of claims 1 to 23, for use in conjunction with exercise therapy.

25. A cell preparation according to any one of claims 1 to 24, for use in subjects experiencing inflammation in muscle tissue.

26. A cell preparation according to any one of claims 1 to 25, for intravenous administration.

27. ​​The cell preparation according to any one of claims 1 to 26, wherein the sarcopenia or frailty is caused by aging.

28. A cell preparation according to any one of claims 1 to 27, for administration to middle-aged and elderly people.

29. A composition containing extracellular vesicles for use in the prevention or treatment of sarcopenia or frailty, The extracellular vesicles mentioned above contain exosomes obtained from the culture supernatant of mesenchymal cells. Composition containing extracellular vesicles.

30. The extracellular vesicle-containing composition according to claim 29, wherein the mesenchymal cells are umbilical cord-derived mesenchymal cells.

31. The extracellular vesicle-containing composition according to claim 29 or 30, wherein the exosome is positive for at least one marker selected from the group consisting of CD9, CD63, and CD81.

32. The extracellular vesicle-containing composition according to any one of claims 29 to 31, wherein the particle size of the exosomes is 100 to 300 nm.

33. The extracellular vesicle-containing composition according to any one of claims 29 to 32, wherein the exosomes are exosomes taken up by muscle cells.

34. The extracellular vesicle-containing composition according to any one of claims 29 to 33, wherein the exosomes suppress apoptosis in muscle cells.

35. The extracellular vesicle-containing composition according to claim 34, wherein the apoptosis is apoptosis induced by H₂O₂.

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