Cell preparation for use in preventing muscle mass loss

Abstract

Provided is a cell preparation capable of preventing a decrease in muscle mass.　The cell preparation according to the present invention, which is to be used for preventing a decrease in muscle mass, comprises umbilical cord-derived cells.

Description

[Technical Field]

[0001] The present invention relates to a cell preparation for use in suppressing loss of muscle mass. [Background technology]

[0002] Japan is rapidly aging, and the number of elderly people is predicted to exceed 30% of the total population by 2025. As the elderly age, they experience impairments in various daily living functions. Age-related sarcopenia (age-related sarcopenia), a condition associated with muscle mass loss due to aging, is known to be one of the causes of these impairments. Sarcopenia is also known as a type of age-related sarcopenia. Sarcopenia is closely associated with unsteadiness, falls, and even frailty in the elderly, which can lead to a state of need for nursing care, creating a problem (Non-Patent Document 1). While improving nutritional status, increasing daily physical activity, and engaging in light exercise have been recommended, these practices are difficult for many elderly people to implement, necessitating the development of new therapeutic interventions. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Masafumi Kuzuya, "3. Diagnosis, pathology, and treatment of sarcopenia," Journal of the Japan Geriatrics Society, Vol. 52, No. 4, pp. 343-349, 2015. Summary of the Invention [Problem to be solved by the invention]

[0004] Exercise and nutritional interventions are currently being implemented as treatments for age-related sarcopenia, but it is unclear whether either method is sufficiently effective.

[0005] Therefore, an object of the present invention is to provide a cell preparation that can suppress the loss of muscle mass. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides a cell preparation used for suppressing a decrease in muscle mass, comprising: The cell preparation comprises umbilical cord-derived cells.

[0007] The present invention provides a cell preparation for use in treating age-related sarcopenia, comprising: The present invention also includes a cell preparation for use in suppressing a decrease in muscle mass. [Effects of the Invention]

[0008] According to the present invention, a decrease in muscle mass can be suppressed. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing changes in body weight in Example 1. [Figure 2] FIG. 2 is a graph showing the change in grip strength per body weight in Example 1. [Figure 3] FIG. 3 is a graph showing endurance in Example 1. [Figure 4] FIG. 4 is a graph showing the measurement results of muscle weight in Example 1. [Figure 5] FIG. 5 is a photograph showing the results of staining muscle tissue in Example 2. [Figure 6] FIG. 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] FIG. 7 is a photograph showing the results of Western blotting in Example 3. [Figure 8] FIG. 8 shows photographs and graphs showing the results of measuring mitochondria in Example 3. [Figure 9] FIG. 9 shows photographs and graphs illustrating the results of apoptosis analysis in Example 4. [Figure 10]FIG. 10 shows a graph showing the expression levels of inflammatory cytokines and a photograph of muscle tissue in Example 5. [Figure 11] FIG. 11 is a graph showing the expression level of TGF-β1 and a photograph of muscle tissue in Example 6. [Figure 12] FIG. 12 is a graph or photograph showing the expression levels of CatK, Sirt1, and MHC in Example 7. [Figure 13] FIG. 13 is an electron microscope photograph of exosomes in Example 8. [Figure 14] FIG. 14 is a photograph of an exosome marker obtained by Western blotting in Example 8. [Figure 15] FIG. 15 is a graph showing the particle size distribution of exosome markers in Example 8. [Figure 16] FIG. 16 is a photograph showing C2C12 cells that have taken up exosomes in Example 8. [Figure 17] FIG. 17 shows photographs and a graph showing apoptosis of C2C12 cells into which exosomes were taken up in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Preparation for preventing muscle mass loss> As described above, the present invention relates to a cell preparation used to suppress muscle mass loss (hereinafter also referred to as a "preparation for suppressing muscle mass loss"), which cell preparation comprises umbilical cord-derived cells. The preparation for suppressing muscle mass loss of the present invention is characterized by containing umbilical cord-derived cells, and other configurations and conditions are not particularly limited. As described below, the umbilical cord-derived cells are presumed to have the functions of improving mitochondrial functionality in muscle, muscle tissue, or muscle cells (also referred to as "muscle fibers (myofibers)" or "muscle cells," hereinafter the same). They also suppress inflammation, apoptosis, muscle cell proliferation, muscle damage, interstitial fibrosis of muscle tissue, and / or muscle repair. Therefore, by containing the umbilical cord-derived cells, the preparation for suppressing muscle mass loss of the present invention can suppress muscle mass loss.

[0011] In the present invention, "muscle mass" refers to, for example, the muscle mass of a subject's limbs, specifically the mass of the gastrocnemius muscle. In the present invention, the "muscle mass" may be evaluated by, for example, measuring the weight of the subject's muscle, or by indirectly calculating the volume using images obtained by dual-energy X-ray absorptiometry (DEXA), bioelectrical impedance spectroscopy, computed tomography (CT), or magnetic resonance imaging (MRI). When the subject is a mammal other than a human, the muscle mass is preferably evaluated by measuring the weight of the subject's muscle. On the other hand, when the subject is a human, the muscle mass is preferably evaluated by indirectly calculating the volume using dual-energy X-ray absorptiometry (DEXA), bioelectrical impedance spectroscopy, or the like.

[0012] In the present invention, the "suppression of muscle mass decline" means that the decline in muscle mass (also referred to as "decrease," "involution," or "reduction," hereinafter the same) is significantly suppressed (also referred to as "inhibition," "block," or "prevention," hereinafter the same). Specifically, the "suppression of muscle mass decline" means, for example, that the degree of muscle mass decline in a subject administered with the cell preparation is significantly suppressed compared to a subject not administered with the cell preparation. Therefore, in the present invention, even if the muscle mass of a subject administered with the cell preparation has decreased compared to when the administration of the cell preparation was started, it can be said that "suppression of muscle mass decline" has occurred as long as the degree of muscle mass decline is significantly suppressed compared to a subject not administered with the cell preparation.

[0013] As shown in the Examples below, the cell preparation of the present invention is presumed to exhibit an inhibitory effect on muscle mass loss by increasing muscle mass. Therefore, the cell preparation of the present invention used to inhibit muscle mass loss can also be referred to as a cell preparation used to increase muscle mass (also referred to as "enhancement," "augmentation," or "bulking"; the same applies hereinafter).

[0014] As shown in the Examples below, the cell preparation of the present invention inhibits a decrease in muscle strength, such as sustained muscle strength, or promotes an increase in said muscle strength. This is presumably due to the fact that the cell preparation of the present invention exhibits a function of inhibiting a decrease in muscle mass. Therefore, the cell preparation of the present invention used to inhibit a decrease in muscle mass can also be referred to as a cell preparation used, for example, to inhibit or increase (also referred to as "increase" or "enhancement"; the same applies hereinafter) muscle strength.

[0015] In the present invention, the term "umbilical cord" refers to a white tubular tissue that connects a fetus and a placenta, and does not contain a placenta or umbilical cord blood. In the present invention, the origin of the "umbilical cord" is not particularly limited, and examples thereof include the umbilical cords of mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions. The umbilical cords are preferably from primates, and more preferably from humans.

[0016] In the present invention, the "umbilical cord" may be an umbilical cord collected from a subject to be administered, treated, or treated (hereinafter collectively referred to as "subject to be administered"), or may be an umbilical cord collected from a subject other than the subject to be administered. From the viewpoint of not being subject to any restrictions during preparation, it is desirable to use an umbilical cord collected from a subject other than the subject to be administered. As shown in the Examples described below, the umbilical cord-derived cells of the present invention may also be cells derived from an umbilical cord collected from a subject other than the subject to be administered. It has been confirmed that the umbilical cord-derived cells exert a therapeutic effect without being eliminated, for example, by immune rejection or the like.

[0017] In the present invention, the umbilical cord can be collected by removing the placenta from postpartum tissue containing the placenta and / or umbilical cord delivered by vaginal delivery or cesarean section. In the present invention, the umbilical cord may be collected from which umbilical cord blood has been removed, and may also be subjected to a sterile or bacteriostatic treatment. The removal of umbilical cord blood can be performed, for example, by rinsing or perfusion with a solution containing an anticoagulant such as heparin. The sterile or bacteriostatic treatment is not particularly limited and can be performed, for example, by application of a disinfectant such as povidone-iodine; immersion in a medium or buffer containing an antibiotic and / or antifungal agent such as penicillin, streptomycin, amphotericin B, gentamicin, and / or nystatin; or the like. Furthermore, the umbilical cord may be subjected to selective lysis of red blood cells, if necessary. The method for selectively lysing the red blood cells can be a method well known in the art, such as lysis with ammonium chloride followed by incubation in a hypertonic or hypotonic medium.

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

[0019] The umbilicus-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 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 genes and / or proteins of IDO (indoleamine 2,3-dioxygenase), PGE2 (prostaglandin E2), and PD-L1 is induced.

[0020] In the present invention, "positive" means that a higher signal or the like is detected by an analytical method such as flow cytometry that utilizes an 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 or the like that is equal to or lower than 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 the present invention, the "HLA-class I" refers to HLA-A, B, or C. In the present invention, the "HLA-class II" refers to HLA-DR, DQ, or DP.

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

[0023] In the present invention, cell preparation extracts and / or secretions of said umbilical cord-derived cells Including The extract of the umbilical cord-derived cells may be, for example, a product obtained by subjecting the umbilical cord-derived cells to concentration treatment, centrifugation treatment, drying treatment, lyophilization treatment, solvent treatment, surfactant treatment, enzymatic treatment using proteases, glycosidases, or the like, protein extraction treatment, ultrasonic treatment, and / or grinding treatment, or a product obtained by a combination of these treatments. The secretory product of the umbilical cord-derived cells may be, for example, exosomes (extracellular vesicles), cell culture supernatant of the umbilical cord-derived cells, and the like.

[0024] In the present invention, "extracellular vesicles" refer to membrane-containing vesicles secreted from cells. Extracellular vesicles are generally thought to be formed in endosomes of the cell of origin and then released outside the cell. Therefore, extracellular vesicles typically comprise a lipid bilayer membrane and a lumen, with the lumen surrounded by the lipid bilayer membrane. Furthermore, the lipid bilayer membrane contains lipids derived from the cell membrane of the cell of origin. The lumen contains cytoplasm derived from the cell of origin. Extracellular vesicles are classified into, for example, exosomes, microvesicles (MVs), apoptotic bodies, etc., based on their size and / or surface markers.

[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 below. The average diameter of the extracellular vesicles can be adjusted, for example, by filtering a liquid containing the extracellular vesicles using a filter having a desired pore size.

[0026] In the present invention, the cell preparation preferably 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 index, in accordance with Example 2 described below, specifically, using the fibrotic area in the interstitium of muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice as an index (interstitial fibrosis assay). In the evaluation, for example, if the interstitial fibrosis area in the group administered with 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 of the interstitial fibrosis area in the group not administered with the test substance, the test substance can be evaluated as having an inhibitory effect on interstitial fibrosis in muscle tissue. For example, in the interstitial fibrosis assay, 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 a group to which the cell preparation was administered. Because the preparation for suppressing muscle mass loss of the present invention can suppress interstitial fibrosis in muscle tissue, it can also be said to be a cell preparation used for suppressing interstitial fibrosis in muscle tissue, for example.

[0027] In the present invention, "muscle tissue" means tissue composed of muscle cells (muscle fibers), for example, excitable tissue having contractility.

[0028] In the present invention, "interstitium" means the space between muscle fibers.

[0029] In the present invention, the cell preparation preferably exhibits an effect of improving mitochondrial function. The effect of improving mitochondrial function can be evaluated, for example, in accordance with Example 3 described below, using the expression level of mitochondrial function-improving 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 an indicator. Specifically, the expression level of mitochondrial function-improving genes in mitochondria in 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 an indicator (mitochondrial function assay). In the evaluation, for example, if the expression level of the mitochondrial function-improving gene in the test substance-administered group is 1.5-fold or more, 2-fold or more, 2.5-fold or more, 5-fold or more, 10-fold or more, 25-fold or more, 50-fold or more, 100-fold or more, 125-fold or more, or 250-fold or more compared to the expression level of the mitochondrial function-improving gene in the test substance-unadministered group, the test substance can be evaluated as having a mitochondrial function-improving effect. Furthermore, in the evaluation, for example, if the number of mitochondria in the test substance-administered group 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 test substance-unadministered group, the test substance can be evaluated as having a mitochondrial function-improving effect. For example, in the mitochondrial function assay, the cell preparation can increase the expression level of the mitochondrial function-improving 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, when compared to a group to which the cell preparation is administered, with the upper limit being, for example, 1000 times or less or 500 times or less.For example, in the mitochondrial function assay, 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, with the upper limit being, for example, 150% or less or 125% or less, when compared to a control group. The preparation for suppressing a decrease in muscle mass of the present invention improves mitochondrial function in muscle tissue, and therefore can be referred to as, for example, a cell preparation used for improving mitochondrial function in muscle tissue. Furthermore, the preparation for suppressing a decrease in muscle mass of the present invention can induce the expression of genes related to mitochondrial functionality in muscle tissue, and therefore can be referred to as, for example, a cell preparation used for inducing (inducing expression of) genes that improve mitochondrial function in muscle tissue. Furthermore, since the preparation for suppressing a decrease in muscle mass of the present invention can increase the number of mitochondria in muscle tissue, it can also be said to be, for example, a cell preparation used to increase the number of mitochondria in muscle tissue.

[0030] For the nucleotide sequences of mRNA and amino acid sequences of proteins of human PGC1-α, human COX4, and human GLUT4, reference can be made to the sequences registered in Genbank under the following accession numbers, for example. 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, the cell preparation preferably exhibits an inhibitory effect on muscle cell apoptosis. The inhibitory effect on muscle cell apoptosis can be evaluated, for example, using the expression level of cleaved-caspase-3 and / or cleaved-caspase-8 or DNA fragmentation (genomic DNA fragmentation) as an index, as described in Example 4 below, specifically, the number of apoptotic cells in muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice as an index (apoptosis assay). In the evaluation, for example, if the number of apoptotic cells in the group administered with 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 with the test substance, the test substance can be evaluated as having an inhibitory effect on muscle cell apoptosis. For example, in the apoptosis assay, 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, compared to a control group. Because the preparation for suppressing muscle mass loss of the present invention inhibits apoptosis in muscle cells, it can also be referred to as a cell preparation used for suppressing apoptosis in muscle cells, for example. The muscle cells are, for example, skeletal muscle cells.

[0032] In the present invention, the cell preparation preferably exhibits anti-inflammatory activity, which can be evaluated, for example, in accordance with Example 5 described below, using as an index the ability to induce inflammatory cytokine genes or chemokine genes, specifically, the expression levels of inflammatory cytokine genes or chemokine genes in muscle tissues (e.g., gastrocnemius or soleus muscles) of SAMP10 mice (inflammation assay). In the evaluation, for example, if the expression level of inflammatory cytokine genes or chemokine genes in the group administered with 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 with the test substance, the test substance can be evaluated as having anti-inflammatory activity. For example, in the inflammation assay, the cell preparation can reduce the expression level of the inflammatory cytokine gene and / or chemokine gene 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 a group to which the cell preparation was administered. The preparation for suppressing muscle mass loss of the present invention exhibits anti-inflammatory activity in muscle tissue, and therefore can also be referred to as, for example, a cell preparation used for suppressing inflammation in muscle tissue. Furthermore, since the preparation for suppressing a decrease in muscle mass 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, it can also be said to be, for example, a cell preparation used for suppressing the expression or induction of expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue.

[0033] For the nucleotide sequences of mRNA and amino acid sequences of proteins of human TNF-α and human MCP-1, reference can be made to the sequences registered in Genbank under the following accession numbers, for example. 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, the cell preparation preferably exhibits a muscle cell proliferation-promoting activity. The proliferation-promoting activity can be evaluated, for example, in accordance with Example 6 described below, using muscle cell proliferation or the expression of a muscle cell proliferation-inducing gene such as TGF (Transforming growth factor)-β1 as an index, specifically, muscle cell proliferation or the expression level of a muscle cell proliferation-inducing gene in muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice as an index (muscle proliferation assay). In the evaluation, for example, if the number of muscle cells in the group administered with 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 with the test substance, the test substance can be evaluated as having the effect of promoting muscle cell proliferation. Furthermore, in the evaluation, for example, if the expression level of the muscle cell proliferation-inducing gene in the group administered with 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, 250 times or more, or 500 times or more compared to the expression level of the muscle cell proliferation-inducing gene in the group not administered with the test substance, the test substance can be evaluated as having a muscle cell proliferation-promoting effect. For example, in the muscle proliferation assay, 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, compared to a group not administered the cell preparation, with the upper limit being, for example, 150% or less or 100% or less.For example, in the muscle proliferation assay, the cell preparation can increase the expression level of the proliferation-inducing gene in the muscle cells by 1.5-fold or more, 2-fold or more, 2.5-fold or more, 5-fold or more, 10-fold or more, 25-fold or more, 50-fold or more, 100-fold or more, 125-fold or more, 250-fold or more, or 500-fold or more, with the upper limit being, for example, 1000-fold or less or 750-fold or less, when compared to a control group. The preparation for suppressing a decrease in muscle mass of the present invention can promote (also referred to as "accelerate," "increase," or "enhance"; the same applies hereinafter) the proliferation of the muscle cells, and therefore can be referred to as, for example, a cell preparation used to promote the proliferation of muscle cells or skeletal muscle of muscle tissue. Furthermore, the preparation for suppressing a decrease in muscle mass of the present invention can induce the expression of the proliferation-inducing gene in the muscle cells, and therefore can be referred to as, for example, a cell preparation used to induce (induce expression of) a proliferation-promoting gene in muscle tissue. The muscle cells are, for example, skeletal muscle cells.

[0035] For the nucleotide sequence of human TGF-β1 mRNA and the amino acid sequence of the protein, reference can be made to the sequences registered in Genbank under the following accession numbers, for example. Human TGF-β1: mRNA accession number: NM_000660 Protein accession number: NP_000651

[0036] In the present invention, the cell preparation preferably promotes repair of muscle tissue, preferably skeletal muscle muscle fiber. The muscle tissue repair ability can be evaluated, for example, according to Example 7 described below, using as an index the expression level of muscle tissue repair-promoting genes, such as Sirt1 (Sirtuin 1) gene and myosin heavy chain (MHC) gene, or the protein expression level of repair-promoting genes, such as Sirt1 and / or myosin heavy chain, specifically, the expression level of muscle tissue repair-promoting genes in muscle tissue (e.g., gastrocnemius or soleus muscle) of SAMP10 mice (muscle repair assay). In the evaluation, for example, if the expression level of muscle tissue repair-promoting genes in the group administered with 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 with the test substance, the test substance can be evaluated as having a muscle tissue repair effect. For example, in the muscle repair assay, the cell preparation can increase the expression level of the muscle tissue repair-promoting gene 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 the upper limit being, for example, 150% or less or 100% or less. The preparation for suppressing muscle mass loss of the present invention exhibits the ability to repair muscle tissue or skeletal muscle, and therefore can also be referred to as, for example, a cell preparation used for repairing muscle tissue or skeletal muscle. Furthermore, since the preparation for suppressing muscle mass loss of the present invention can induce the expression of a repair-promoting gene or the expression of a protein of a repair-promoting gene in muscle tissue or skeletal muscle, it can also be referred to as a cell preparation used for promoting the repair of muscle tissue or a cell preparation used for inducing the expression of a repair-promoting gene in muscle tissue. The muscle tissue is, for example, skeletal muscle tissue.

[0037] For the nucleotide sequences of mRNA and amino acid sequences of proteins of human SIRT1 and MHC, reference can be made to, for example, the sequences registered in Genbank under the following 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, preferably skeletal muscle. The ability to suppress muscle tissue damage can be evaluated, for example, using the expression level of a muscle damage-inducing gene, such as the cathepsin K (CatK, Cathepsin K, CTSK) gene, as an index, according to Example 7 described below. CatK causes muscle damage, for example, by inducing the degradation of muscle cells. Since the preparation for suppressing a decrease in muscle mass of the present invention exhibits the ability to suppress damage to muscle tissue or skeletal muscle, it can also be referred to as, for example, a cell preparation used for suppressing damage to muscle tissue or skeletal muscle. Furthermore, since the preparation for suppressing a decrease in muscle mass of the present invention can suppress the expression of a muscle damage-inducing gene in muscle tissue or skeletal muscle, it can also be referred to as a cell preparation used for suppressing the expression of a muscle damage-inducing gene. The muscle tissue is, for example, skeletal muscle tissue.

[0039] For the nucleotide sequence of human CatK mRNA and the amino acid sequence of the protein, see, for example, the sequences registered in Genbank under the following 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, a step of passaging 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 passaging. Another example of the preparation method includes, for example, (A) a step of dissociating the tissue by cutting the umbilical cord or by enzymatic treatment, or both, (B) a step of culturing the umbilical cord tissue, and (C) a step of passaging. The umbilical cord-derived cells may be a homogeneous cell population or a heterogeneous cell population.

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

[0042] First, a method including the steps (1) to (3) will be described. The step (1) of cutting the umbilical cord can be carried out, for example, by cutting the umbilical cord obtained by the above-mentioned method using mechanical force (chopping force or shearing force) while the cord contains the amniotic membrane, blood vessels, perivascular tissue, and / or Wharton's jelly. The size of the umbilical cord section obtained by cutting is not particularly limited, and may be, for example, 1 to 10 mm. 3 , 1~5mm 3 , 1 to 4 mm 3 , 1~3mm 3 or 1 to 2 mm 3 Examples include:

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

[0044] In the present invention, the "culture vessel" may be, for example, a culture vessel having a solid surface. Examples of the culture vessel that can be used include culture vessels used for culturing cells, tissues, and / or organs. The "solid surface" refers to, for example, any material that allows binding to the umbilical cord-derived cells. Specific examples of the material include plastic materials that have been treated to promote binding of mammalian cells to their surface (e.g., treatment to increase hydrophilicity). The type of culture vessel having a solid surface is not particularly limited, and examples include petri dishes, dishes, flasks, etc.

[0045] In the present invention, the "culture medium suitable for umbilical cord-derived cells" can be prepared, for example, by adding additives such as serum to a basal medium. Examples of the 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 further contain, as needed, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antifungals, antioxidants, pyruvic acid, buffers, and inorganic salts. The basal medium is not particularly limited, and examples thereof include Dulbecco's Modified Eagle's Medium (DMEM) (high glucose or low glucose), improved DMEM, DMEM / MCDB 201, Eagle's basal 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 mixtures thereof. Examples of the serum include animal serum such as human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, and rat serum. The amount of serum added to the basal medium is, for example, 5% to 15% (v / v), preferably about 10% (v / v). The fatty acids are not particularly limited, and examples thereof include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. The lipids are not particularly limited, and examples thereof include phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. The amino acids are not particularly limited, and examples thereof include L-isomers 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, as well as D-isomers thereof and mixtures thereof (DL-isomers).The protein is not particularly limited, and examples thereof include ecotin, reduced glutathione, fibronectin, β2-microglobulin, etc. The polysaccharide is not particularly limited, and examples thereof include glycosaminoglycans such as hyaluronic acid and heparan sulfate, etc. The growth factor is not particularly limited, and examples thereof include 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 inhibitory factor (LIF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), erythropoietin (EPO), etc. The antibiotic and / or antifungal agent is not particularly limited, and examples thereof include penicillin G, streptomycin sulfate, amphotericin B, gentamicin, nystatin, and mixtures thereof.

[0046] In step (2), the seeded umbilical cord sections are preferably held down using a plate or the like during the culture period to prevent them from floating in the culture medium. Examples of the plate include the plate described in JP 2015-70824 A.

[0047] In step (2), the culture conditions are not particularly limited, and reference can be made to general culture conditions for cells, tissues, organs, etc. Specific examples include a CO2 concentration of 0 to 5% in step (2), an O2 concentration of 2 to 25%, and preferably 5 to 20% in step (2), and a culture temperature of 25 to 40°C, and preferably about 37°C (35 to 39°C).

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

[0049] In step (2), for example, after the culturing, the cells are washed to remove unbound cells and cell debris, and then detached using a detachment agent containing a solution containing a chelating agent such as EDTA, a protease such as trypsin, collagenase, or dispase, a glycolytic enzyme such as hyaluronidase, or a mixture thereof. In step (2), for example, the detachment solution containing the cells and the umbilical cord section is 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, in the aforementioned culture vessel and cultured using the aforementioned culture medium.

[0050] In the step (3), the umbilical cord-derived cells can be proliferated to a required number by subculturing. In the step (3), the cells may be detached using the detachment agent in the subculturing, and then seeded at an appropriate cell density in a separately prepared culture vessel for continued culture. The cell density (seeding density) when seeding the cells may be, 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 and the like, and preferably 2 × 10 3 ~1×10 4 cells / cm 2 The seeding density is preferably adjusted so that it takes 3 to 7 days to reach an appropriate confluency. In the subculture in the step (3), the medium may be replaced as needed.

[0051] The number of passages in step (3) is not particularly limited and may be, for example, until senescence, at which point cell division ceases. From the viewpoint of therapeutic use, the number of passages in step (3) is preferably 3 to 25, and more preferably 4 to 12.

[0052] Next, a method including steps (A) to (C) will be described. In step (A), the enzymatic treatment is carried out by enzymatically treating the umbilical cord obtained by the method described above with the amniotic membrane, blood vessels, perivascular tissue, and / or Wharton's jelly to dissociate the tissues. The enzyme used in the enzymatic treatment is not particularly limited, and examples thereof include proteases such as collagenase and dispase; glycolytic enzymes such as hyaluronidase; and the like.

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

[0054] In this way, the umbilical cord-derived cells can be obtained after the step (3) or (C).

[0055] To confirm that the cells obtained by the method for preparing umbilical cord-derived cells are the umbilical cord-derived cells, they may be analyzed for surface antigens by conventional methods such as flow cytometry. Furthermore, the cells obtained by the method for preparing umbilical cord-derived cells may be evaluated for their identity by measuring the amounts of various proteins produced by the cells.

[0056] The cells obtained by the method for preparing umbilical cord-derived cells may be prepared for therapeutic use as is, or may be cryopreserved. The cryopreservation is carried out, for example, by suspending the cells in a cryopreservation solution suitable for preserving the umbilical cord-derived cells and storing them at −80°C to −180°C. The cryopreservation solution is not particularly limited, and examples thereof include aqueous solutions 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 are preferably DMSO and / or propylene glycol, and more preferably DMSO. The cryoprotectant is contained in the cryopreservation solution at, 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 cryoprotectant is contained in the cryopreservation solution at, for example, 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 content in the cryopreservation solution is, for example, 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 such components include a pH adjuster and a thickener. Examples of such pH adjusters include sodium bicarbonate, HEPES, and phosphate buffer. Furthermore, when a phosphate buffer is not added to the basic stock solution (BSS), the pH adjuster may be a solution containing, for example, sodium chloride, which provides buffering capacity at a pH near the pH suitable for the umbilical cord-derived cells. A phosphate buffer is preferably used as the pH adjuster. The pH adjuster is preferably used 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, the "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, or potassium dihydrogen phosphate (anhydrous). A buffer containing sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous) is particularly preferred. The pH adjuster is contained in the cryopreservation solution in an amount of, for example, 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 the natural animal-derived components include the serum and basal medium described above. Preferably, the cryopreservation solution does not contain natural animal-derived components. The cryopreservation solution free of natural animal-derived components does not cause quality differences between lots of natural animal-derived components, can suppress the possibility of changes in the properties of cells in umbilical cord tissue due to components such as various cytokines, growth factors, and hormones contained in serum, and can also suppress the effects of components of unknown origin contained in the basal medium. For this reason, the cryopreservation solution free of natural animal-derived components is extremely useful, especially for clinical use.

[0060] The cryopreservation solution may further contain a thickener. The thickener is not particularly limited, and examples thereof include those capable of constituting a cryopreservation solution capable of adequately preserving the umbilical cord tissue. Examples of the thickener include carboxymethylcellulose (hereinafter also referred to as "CMC"), carboxymethylcellulose sodium (hereinafter also referred to as "CMC-Na"), organic acid polymers, propylene glycol alginate, and sodium alginate. Preferred thickeners are CMC and CMC-Na, with CMC-Na being particularly preferred. The organic acid polymer is preferably sodium polyacrylate. The thickener is contained in the cryopreservation solution, for example, at 0.1 to 1 w / v %, preferably 0.1 to 0.5 w / v %, and more preferably 0.2 to 0.4 w / v %.

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

[0062] The cryopreservation solution is preferably an aqueous solution containing a thickener, a cryoprotectant, and glucose, and is free of natural animal-derived components. The cryopreservation solution is more preferably an aqueous solution containing CMC-Na, DMSO, and glucose, and is free of natural animal-derived components. The cryopreservation solution is even more preferably 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 is free of natural animal-derived components.

[0063] The cells obtained by the umbilical cord-derived cell preparation method may be used as a cell preparation for various applications, for example, by mixing with an infusion preparation. Furthermore, when the umbilical cord-derived cells are cryopreserved, the cryopreserved umbilical cord-derived cells may be suspended in the cryopreservation solution and thawed and then used as a cell preparation for various applications. Alternatively, the thawed cells may be mixed with an infusion preparation, and the resulting mixture may be used as a cell preparation for various applications. When mixing with the infusion preparation, the culture medium or cryopreservation solution in which the umbilical cord-derived cells are suspended may be mixed with the infusion preparation, or the cells may be separated from the solvent by centrifugation or the like, and then the cells alone may be mixed with the infusion preparation. To avoid the complexity of the procedure, the preparation method preferably does not include a step of culturing the frozen cells after thawing, or the cryopreservation solution in which the thawed cells are suspended may be directly mixed with the infusion preparation.

[0064] In the present invention, the "infusion preparation" includes, for example, solutions such as infusions used in human treatment, and specific examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, No. 1 solution, No. 2 solution, No. 3 solution, No. 4 solution, etc.

[0065] The preparation for suppressing a decrease in muscle mass of the present invention may be a kit containing the infusion preparation in addition to the umbilicus-derived cells.

[0066] The preparation for suppressing muscle mass loss of the present invention may contain, in addition to or instead of the infusion preparation, a pharmaceutically acceptable carrier, such as a suspending agent, solubilizer, stabilizer, isotonicity agent, preservative, anti-adsorption agent, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc., for administering the cell preparation, and may be added appropriately within a range that does not impair the effects of the present invention.

[0067] The suspending agent is not particularly limited, and examples thereof include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, powdered tragacanth, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0068] The solution adjuvant is not particularly limited, and examples thereof include 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 thereof include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.

[0070] The tonicity agent is not particularly limited, and examples thereof include D-mannitol, sorbitol, and the like.

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

[0072] The adsorption inhibitor is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, and polyethylene glycol.

[0073] The sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thiochitic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

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

[0075] The preparation for suppressing a decrease in muscle mass of the present invention may further contain, as appropriate, commonly added ingredients 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. Furthermore, for example, ACD-A solution (composed of sodium citrate hydrate, citric acid hydrate, glucose, etc.) may be added as an anticoagulant and / or the pH adjuster.

[0076] The preparation for inhibiting muscle mass loss of the present invention may be mixed with, for example, organic substances such as biopolymers; inorganic substances such as hydroxyapatite; etc. for local administration, and as specific examples, it may be mixed with a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and chemical derivatives thereof.

[0077] The preparation for suppressing a decrease in muscle mass of the present invention can be, for example, in In vitro It can also be used in in vivo The preparation for suppressing muscle mass loss of the present invention can be used, for example, as a research reagent or as a pharmaceutical. In the latter case, the preparation for suppressing muscle mass loss of the present invention can also be referred to as a cell preparation or a pharmaceutical cell preparation used in the treatment of age-related sarcopenia.

[0078] There are no particular limitations on the subjects to which the preparation for suppressing muscle mass loss of the present invention is administered. in vivo When used in the above, the subject of administration can be, for example, a human or a non-human animal other than a human. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions; birds; and fish. in In vitro When used in the above, the subject of administration can be, for example, a cell, tissue, organ, etc., and examples of the cells can be, for example, cells collected from a living body or cultured cells, and examples of the tissue or organ can be, for example, tissue (biological tissue) or organ collected from a living body, etc. Examples of the cells can be, for example, muscle cells, iPS cells (induced pluripotent stem cells), stem cells, etc.

[0079] The preparation of the present invention for suppressing muscle mass loss in vivo When used in the above, the subject of administration may be a subject in which inflammation is occurring in muscle tissue, a subject in which induction of inflammatory cytokine gene expression (also referred to as "enhancement," "increase," "improvement," or "enhancement," hereinafter) and / or induction of chemokine gene expression is occurring in muscle tissue, a subject in which interstitial fibrosis is induced or occurring in muscle tissue, a subject in which suppression of muscle tissue repair-promoting gene expression is occurring, a subject in which mitochondrial function is decreasing in muscle cells, a subject in which the number of mitochondria is decreasing in muscle cells, a subject in which muscle tissue is damaged, a subject in which induction of muscle tissue damage-inducing gene expression is occurring, and / or a subject in which apoptosis of muscle cells is increasing (enhancement).

[0080] The preparation of the present invention for suppressing muscle mass loss in vivoWhen used in the above, the subject is preferably a subject experiencing a decrease in muscle mass. Furthermore, in the preparation for inhibiting a decrease in muscle mass of the present invention, the subject is preferably a subject experiencing a decrease in muscle mass due to aging, since the decrease in muscle mass in age-related sarcopenia can be inhibited, as described below. Furthermore, age-related sarcopenia occurs, for example, in middle-aged and elderly people. Therefore, the subject is preferably middle-aged and elderly. 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 a decrease in muscle mass of the present invention are not particularly limited, and the administration form, administration time, administration amount, etc. can be appropriately set depending on the type of subject to be administered, etc.

[0082] The method of administration of the preparation for suppressing a decrease in muscle mass of the present invention can be exemplified by, for example, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intravenous administration, etc., but intravenous administration is preferred because it can be administered safely and stably regardless of the skill of the administerer.

[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, can provide an inhibitory effect on muscle mass loss (therapeutic effect) against a disease compared to a subject not administered the preparation. Specifically, the dosage can be appropriately determined depending on, for example, the age, weight, symptoms, etc. of the subject. Specifically, the dosage is, 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 Preferably, 10 4 ~10 8 pieces / kg weight, 10 4 ~10 7 The dose is, for example, 10 cells / kg body weight as the number of umbilical cord-derived cells per administration. 4 ~10 9 pieces, 10 6 ~109 pieces, 10 4 ~10 8 pieces, 10 4 ~10 7 Preferably, 10 4 ~10 8 pieces, 10 4 ~10 7 The dose can also be expressed as, 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 may be 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 appropriately determined while confirming the therapeutic effect on the subject. When administering multiple times, the administration interval may be appropriately determined while confirming the therapeutic effect on the subject, and may be, for example, once a day, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0085] The preparation for suppressing muscle mass loss of the present invention may be used in combination with, for example, other drugs and / or methods used to suppress muscle mass loss, such as exercise therapy such as resistance exercise, and specific examples thereof can be found in the following References 1 to 3. Reference 1: Hidetaka Wakabayashi, "Actual Practice of Exercise Therapy for Sarcopenia," 2013, Japan Medical News, 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 loss of the present invention can be suitably used in subjects experiencing muscle mass loss, specifically, in subjects with age-related sarcopenia. Age-related sarcopenia refers to, for example, a condition or disease in which muscle mass, such as skeletal muscle, decreases with age. Examples of age-related sarcopenia 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. Therefore, the present invention may 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, in which the cell preparation for suppressing muscle mass loss of the present invention is used in the subject. The method for suppressing muscle mass loss of the present invention includes, for example, a step of administering the cell preparation for suppressing muscle mass loss of the present invention to the subject. The administration conditions in the administration step can be as described above.

[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 provides a method for treating a subject experiencing muscle mass loss, in which the cell preparation for suppressing muscle mass loss of the present invention is used in the subject. The method for treating a subject of the present invention includes, for example, a step of administering the cell preparation for suppressing muscle mass loss of the present invention to the subject. The administration conditions in the administration step can be as described above.

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

[0090] In the present invention, "treatment" may mean any of suppressing, preventing, inhibiting, preventing, or delaying the onset of a target disease, stopping, suppressing, inhibiting, or delaying the progression of an already developed target disease or its symptoms, and improving or remitting a target disease.

[0091] <Uses of cell preparations> In another embodiment, the present invention provides a cell preparation for use in improving mitochondrial function in muscle tissue or a method for improving mitochondrial function in muscle tissue. In this case, the present invention provides a cell preparation for use in improving mitochondrial function in muscle tissue, wherein the cell preparation comprises umbilical cord-derived cells. Also, the present invention provides a method for improving mitochondrial function in muscle tissue of a subject, wherein the cell preparation for improving mitochondrial function in muscle tissue of the present invention is used in the subject. The same explanation as for the cell preparation for use in inhibiting muscle mass loss of the present invention can be applied 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 provides a cell preparation for use in increasing the number of mitochondria in muscle tissue, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for increasing the number of mitochondria in muscle tissue of a subject, wherein the cell preparation for increasing the number of mitochondria in muscle tissue is used in the subject. The same explanation as for the cell preparation for use in inhibiting muscle mass loss of the present invention can be applied to the present invention.

[0093] In another embodiment, the present invention provides a cell preparation for use in inducing expression of a gene that improves mitochondrial function in muscle tissue or a method for inducing expression of a gene that improves mitochondrial function in muscle tissue. In this case, the present invention provides a cell preparation for use in inducing expression of a gene that improves mitochondrial function in muscle tissue, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for inducing expression of a gene that improves mitochondrial function in muscle tissue of a subject, wherein the cell preparation for use in inducing expression of a gene that improves mitochondrial function in muscle tissue is used in the subject. The same explanation as for the cell preparation for use in inhibiting muscle mass loss of the present invention can be applied to the present invention.

[0094] In another embodiment, the present invention provides a cell preparation for use in inhibiting apoptosis in muscle cells or a method for inhibiting apoptosis in muscle cells. In this case, the present invention provides a cell preparation for use in inhibiting apoptosis in muscle cells, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for inhibiting apoptosis in muscle cells in a subject, wherein the cell preparation for inhibiting apoptosis in muscle cells is used in the subject. The same explanation as for the cell preparation for inhibiting muscle mass loss of the present invention can be applied to 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 provides a cell preparation for use in anti-inflammation (inflammation suppression) in muscle tissue, wherein the cell preparation comprises umbilicus-derived cells. The present invention also provides a method for suppressing inflammation in muscle tissue in a subject, wherein the cell preparation for use in suppressing inflammation in muscle tissue is used in the subject. The same explanation as for the cell preparation for use in suppressing muscle mass loss of the present invention can be applied to the present invention.

[0096] In another embodiment, the present invention provides a cell preparation used for 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 provides a cell preparation used for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue of a subject, wherein the cell preparation used for suppressing the expression of inflammatory cytokine genes and / or chemokine genes in muscle tissue is used in the subject. The same explanation as for the cell preparation used to suppress muscle mass loss of the present invention can be applied to the present invention.

[0097] In another embodiment, the present invention provides a cell preparation for use in promoting muscle cell proliferation or a method for promoting muscle cell proliferation. In this case, the present invention provides a cell preparation for use in promoting muscle cell proliferation, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for promoting muscle cell proliferation in a subject, wherein the cell preparation for promoting muscle cell proliferation is used in the subject. The same explanation as for the cell preparation for use in inhibiting muscle mass loss of the present invention can be applied to the present invention.

[0098] In another embodiment, the present invention provides a cell preparation for use in inducing expression of a proliferation-promoting gene in muscle cells or a method for inducing expression of a proliferation-promoting gene in muscle cells. In this case, the present invention provides a cell preparation for use in inducing expression of a proliferation-promoting gene in muscle cells, the cell preparation comprising umbilical cord-derived cells. The present invention also provides a method for inducing expression of a proliferation-promoting gene in muscle cells of a subject, comprising: The present invention uses a cell preparation used for inducing the expression of a gene that promotes muscle cell proliferation. The same explanation as for the cell preparation used to suppress a decrease in muscle mass of the present invention can be applied to the present invention.

[0099] In another embodiment, the present invention provides a cell preparation for use in muscle tissue repair or a method for repairing muscle tissue. In this case, the present invention provides a cell preparation for use in muscle tissue repair, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for repairing muscle tissue in a subject, wherein the cell preparation for use in muscle tissue repair is used in the subject. The same explanation as for the cell preparation for use in inhibiting muscle mass loss of the present invention can be applied to the present invention.

[0100] In another embodiment, the present invention provides a cell preparation used for inducing expression of muscle tissue repair genes or a method for inducing expression of muscle tissue repair genes. In this case, the present invention provides a cell preparation used for inducing expression of muscle tissue repair genes, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for inducing expression of muscle tissue repair genes in a subject, wherein the cell preparation used for inducing expression of muscle tissue repair genes is used in the subject. The same explanation as for the cell preparation used to inhibit loss of muscle mass of the present invention can be applied to the present invention.

[0101] In another embodiment, the present invention provides a cell preparation for use in suppressing muscle tissue damage or a method for suppressing muscle tissue damage. In this case, the present invention provides a cell preparation for use in suppressing muscle tissue damage, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for suppressing muscle tissue damage in a subject, wherein the cell preparation for use in suppressing muscle tissue damage is used in the subject. The same explanation as for the cell preparation for use in suppressing muscle mass loss of the present invention can be applied to the present invention.

[0102] In another embodiment, the present invention provides a cell preparation used for suppressing the expression of muscle tissue damage-inducing genes or a method for suppressing the expression of muscle tissue damage-inducing genes. In this case, the present invention provides a cell preparation used for suppressing the expression of muscle tissue damage-inducing genes, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for suppressing the expression of muscle tissue damage-inducing genes in a subject, wherein the cell preparation used for suppressing the expression of muscle tissue damage-inducing genes is used in the subject. The same explanation as for the cell preparation used to suppress muscle mass loss of the present invention can be applied to the present invention.

[0103] In another embodiment, the present invention provides a cell preparation for use in inhibiting interstitial fibrosis in muscle tissue of a subject or a method for inhibiting interstitial fibrosis in muscle tissue. In this case, the present invention provides a cell preparation for use in inhibiting interstitial fibrosis in muscle tissue of a subject, wherein the cell preparation comprises umbilical cord-derived cells. The present invention also provides a method for inhibiting interstitial fibrosis in muscle tissue of a subject, wherein the cell preparation for inhibiting interstitial fibrosis in muscle tissue of a subject is used in the subject. The same explanation as for the cell preparation for inhibiting muscle mass loss of the present invention can be applied to the present invention.

[0104] <Use of cell preparations> The present invention provides a cell composition for use in inhibiting loss of muscle mass, treating age-related sarcopenia, 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, inhibiting apoptosis in muscle cells, inhibiting inflammation in muscle tissue, inhibiting 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 repair genes in muscle tissue, inhibiting muscle tissue damage, and / or inhibiting the expression of muscle tissue damage-inducing genes, wherein the cell preparation comprises umbilical cord-derived cells. The present invention relates to the use of umbilical cord-derived cells for producing a cell composition for use in suppressing muscle mass loss, treating age-related sarcopenia, 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 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 same explanation as for the preparation for suppressing muscle mass loss of the present invention can be applied to the present invention. [Example]

[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, kits, etc. were used according to their protocols.

[0106] [Example 1] It was confirmed that sarcopenia can be treated with the cell preparation 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, after approval by the Ethics Committee of the Institute of Medical Science, The University of Tokyo, and with the donor's consent, all tissue elements of the umbilical cord (including the amnion, blood vessels, perivascular tissue, and Wharton's jelly) were collected and separated into 1-2 mm pieces. 3 The umbilical cord-derived cells were obtained by a modified explant method in which the umbilical cord was cut into small pieces and seeded onto a culture dish. The pieces were then covered with a Cell Amigo (Tsubakimoto Chain Co., Ltd.) and cultured in α-minimal essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cells adhere to plastic.

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

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

[0110] (3) Preparation of cell preparations The obtained umbilical cord-derived cells were seeded onto Corning® CellBIND® surface 100 mm dishes (Corning, Product Number: #3292) and passaged and expanded from passage 1 to passage 4. The culture medium used during the passages was CiMS™-BM (Nipro Corporation, Product Code: 87-070), a basal culture medium for human mesenchymal stem cells, supplemented with CiMS™-sAF (Nipro Corporation, Product Code: 87-072), an animal-free additive for basal culture medium for human mesenchymal stem cells, at the ratio specified in the package insert. The cells were then detached using TrypLE™ Select Enzyme (1X), no phenol red (ThermoFisher, Product Code: 12563011), and collected at 1 x 10 6 The solution was adjusted to a concentration of 150 μl of cells, and used as the cell preparation of the present invention.

[0111] (4) Model animals The animals used in the study were SAMP10 (purchased from Chubu Scientific Materials Co., Ltd.), a strain of SAMP (Senescence-Accelerated Mouse Prone) mice that exhibit accelerated aging and a short lifespan. SAMP10 is a strain that exhibits traits that lead to the early onset of age-related disorders such as learning and memory impairment, emotional disorders, and senile amyloidosis, accompanied by brain atrophy. SAMP10 mice have a short lifespan of approximately one year, and aging symptoms can be observed from around 24 weeks of age, so they were used as a model animal for sarcopenia.

[0112] (5) Preparation of mice 24-week-old SAMP10 mice were randomly selected and the cells were inoculated into a BD Rhodoze™ 30G syringe with a needle for subcutaneous insulin injection (Becton Dickinson, Catalog No. 326638) at a dose of 1 × 10 6 The mice were administered with the same volume of saline or culture medium into the tail vein of mice at 24 weeks of age as controls.

[0113] The mice of the example and the control mice were each divided into a training group (Ex) and a non-training group. Specifically, the group administered with cells but not training (Cell) was designated Example A (n=8), and the group administered with cells and training (Cell+Ex) was designated Example B (n=8). The group not administered with cells but not training was designated Control (n=7), and the group not administered with cells but training (Ex) was designated Reference Example (n=7). The training consisted of treadmill training performed three times a week. The treadmill training involved forcing the mice to run using a training device (a mouse treadmill, manufactured by Melquest Co., Ltd.) equipped with a conveyor belt and a device that generates electrical stimuli. The training content was varied depending on the age of the mice. Specifically, mice aged 24 to 26 weeks were given a 0° incline and warmed up for 5 minutes at 7 m / min, followed by 35 minutes at 17 m / min and a 5-minute cool-down at 7 m / min. Mice aged 27 to 36 weeks were given a 5° incline and warmed up for 5 minutes at 10 m / min, followed by 35 minutes at 18 m / min and a 5-minute cool-down at 10 m / min.

[0114] (6) Results The body weights of the mice in each group were measured at 24, 28, 32, and 36 weeks of age. The results are shown in Figure 1. Figure 1 is a graph showing changes in body weight. In each graph in Figure 1, the vertical axis shows the average body weight (g) of the mice in each group, and the horizontal axis shows 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 (●) plots indicate the control, the square (□) plots indicate Example A (Cell), the triangle (▲) plots indicate the reference example (Ex), and the triangle (▽) plots indicate Example B (Ex+Cell). In addition, 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 mice in 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 mice in each group was measured at 24, 28, 32, and 26 weeks of age. Grip strength measurements were performed 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 portion of the device and allowed to grasp the mesh with its 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 measured immediately before the mouse released the mesh was recorded as grip strength. These results are shown in Figure 2.

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

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

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

[0119] (How to measure endurance) speed slope time Start 6m / min 0° 0~ 5min 6m / min 10° 5~7min 8m / min 10° 7~ 9min 10m / min 10° 9~11min 12m / min 10° 11~13min 14m / min 10° 13~15min 16m / min 10° 15~17min 18m / min 10° 17~19min 20m / min 10° 19~21min 21m / min 10° 21min~ First, each mouse was allowed to run for 5 minutes at a speed of 6 m / min with the device inclined at 0°. After 5 minutes, the inclination was changed to 10°, and the mouse was allowed to run for 2 minutes at 6 m / min with the device inclined at 10°. After that, the speed was increased by 2 m / min every 2 minutes. The maximum speed was set to 21 m / min, and the time until each mouse could no longer 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 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 mice in each group, with the circle (●) plot showing the control, the square (□) plot showing Example A (Cell), the triangle (▲) plot showing the reference example (Ex), and the triangle (▽) plot showing Example B (Ex+Cell). Figure 3(B) is a graph showing the endurance of control mice, Figure 3(C) is a graph showing the endurance of Example A (Cell), Figure 3(D) is a graph showing the endurance of Reference Example (Ex), and Figure 3(E) is a graph showing the endurance of Example B (Ex+Cell).

[0121] As shown in Figure 3, the mice of Example A had significantly improved endurance at 32 weeks of age compared to the control mice, and had even more improved endurance at 36 weeks of age. Furthermore, the mice of Example B had significantly improved endurance at 32 weeks of age compared to the mice of Reference Example (Ex) and the control mice, and had even more improved endurance at 36 weeks of age. Therefore, it was found that the cell preparation of the present invention has an endurance-improving effect, i.e., a therapeutic effect on muscle weakness in age-related sarcopenia.

[0122] [Example 2] It was confirmed that 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, and the weights were measured and the morphology was observed. These results are shown in FIG.

[0124] FIG. 4 is a graph showing the results of measuring muscle weight. FIG. 4(A) is a graph showing the weight of the gastrocnemius muscle of each mouse, and FIG. 4(B) is a graph showing the weight of the soleus muscle of each mouse. In FIG. 4, the vertical axis shows the relative value of muscle mass per mouse body weight (muscle mass (mg) / body weight (g)), and the horizontal axis shows the type of mouse. As shown in FIG. 4, the mice of Example A had significantly increased gastrocnemius and soleus muscle weights 12 weeks after cell administration compared to the control mice. The mice of Example B had significantly increased gastrocnemius and soleus muscle weights 12 weeks after cell administration compared to the control mice.

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

[0126] Figure 5 is a photograph showing the results of muscle tissue staining. In Figure 5, (A) and (B) show the results of gastrocnemius muscle, with (A) showing the results of HE staining and (B) showing the results of MT staining. In Figure 5, (C) and (D) show the results of soleus muscle, with (C) showing the results of HE staining and (D) showing the results of MT staining. In Figures 5(B) and (D), the areas stained blue by MT staining, i.e., the areas of interstitial fibrosis, are enclosed by dashed lines. As shown in Figure 5, 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 the control mice. The mice of Example B showed an increase in muscle fiber size in the gastrocnemius and soleus muscles 12 weeks after cell administration compared to the control mice. Furthermore, compared to control mice, the mice of Examples A and B showed reduced interstitial fibrosis in both the gastrocnemius and soleus muscles. It is known that patients with age-related sarcopenia, such as sarcopenia and frailty, experience a decrease in muscle cross-sectional area (muscle fiber size), interstitial fibrosis, and other conditions. Because the cell preparation of the present invention can increase muscle cross-sectional area and inhibit interstitial fibrosis, it is presumed to be effective in treating age-related sarcopenia.

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

[0128] [Example 3] It was confirmed that administration of the cell preparation of the present invention has the effect of improving mitochondrial function.

[0129] The expression levels of mRNA encoding PGC1-α, COX4, and GLUT4 in the gastrocnemius and soleus muscles of each mouse collected in Example 2 were determined by RT-PCR using a qPCR kit (Powers SYBR® Green CR Master Mix, ThermoFisher, Cat. No. #437659). The expression levels of PGC1-α in the gastrocnemius muscles were also determined by Western blotting. The expression levels of PGC1-α were calculated from the stained gel images. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an endogenous control for RT-PCR and Western blotting (the same applies below). These results are shown in Figures 6 and 7. PGC1-α primer set Forward primer: 5'-CCGAGAATTCATGGAGCAAT-3' (SEQ ID NO: 1) Reverse primer: 5'-TTTCTGTGGGTTTGGTGTGA-3' (SEQ ID NO: 2) COX4 primer set Forward primer: 5'-AGCTGAGCCAAGCAGAGAAG-3' (SEQ ID NO: 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 is a graph showing the expression levels of PGC1-α, COX4, and GLUT4 in the gastrocnemius muscle of each mouse. In Figure 6, the top three graphs are graphs showing the results in the gastrocnemius muscle, and the bottom three graphs are graphs showing the results in the soleus muscle. The top and bottom graphs in Figure 6 show, from left to right, the results for PGC1-α, COX4, and GLUT4. In each graph in Figure 6, the vertical axis indicates the relative expression level of PGC1-α, COX4, or GLUT4 mRNA to the endogenous control (GAPDH), and the horizontal axis indicates the type of mouse.

[0131] Figure 7 is a photograph showing the results of Western blotting. In Figure 7, (A) is a gel image showing the results of 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 row shows the expression level of PGC1-α, and the lower row shows the expression level of the endogenous control (GAPDH). In addition, in the gel image of Figure 7(A), every third lane from the left shows the results of the control (Control), Example A (Cell), Reference Example (Ex), and Example B (Cell+Ex), respectively.

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

[0133] As shown in Figures 7(A) and (B), the mice of Example A showed increased PGC1-α protein expression in the gastrocnemius and soleus muscles 12 weeks after cell administration compared to the control mice. Furthermore, the mice of Example B showed increased PGC1-α protein expression in the gastrocnemius muscles 12 weeks after cell administration compared to the control and reference mice. These results suggest that the cell preparation of the present invention inhibits 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, manufactured by JEOL Ltd.), and the number of mitochondria per unit area was counted. These results are shown in Figure 8.

[0135] Figure 8 shows photographs and diagrams illustrating the results of mitochondrial measurement. In Figure 8, (A) shows the results of an electron microscope image 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 to the control and reference example mice.

[0136] It has been suggested that mitochondrial dysfunction is involved in age-related sarcopenia, frailty, etc. Therefore, it is presumed that the cell preparation of the present invention exerts a therapeutic effect on age-related sarcopenia through the effect of improving mitochondrial function.

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

[0138] The gastrocnemius and soleus muscles of each mouse collected in Example 2 were analyzed by TUNEL (T-mediated digoxygenin (biotin)-dUTP nick end labeling) to determine the amount of fragmented DNA, which indicates cellular apoptosis. Furthermore, the expression levels of cleaved-caspase-3 and cleaved-caspase-8, which are markers of intrinsic apoptosis, were determined 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) is a photograph showing the results of TUNEL staining. The four panels on the left show the results for the gastrocnemius muscle, and the four panels on the right show the results for the soleus muscle. In the four panels on the left and right of Figure 9(A), the upper left panel shows the results for the control (Control), the upper right panel shows the results for Example A (Cell), the lower left panel shows the results for the reference example (Ex), and the lower right panel shows the results for Example B (Cell+Ex). In Figure 9(A), the 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 of Figures 9(B) and (C), the vertical axis indicates the relative expression level of cleaved-caspase-3 or cleaved-caspase-8 to the endogenous control (GAPDH), and the horizontal axis indicates the type of mouse. As shown in Figure 9, the mice of Examples A and B showed a decrease in fragmented DNA, indicating cellular apoptosis, in each muscle tissue at 12 weeks after cell administration compared to the control mice. Furthermore, the mice of Examples A and B showed a decrease in the expression levels of cleaved-caspase-3 and cleaved-caspase-8, which are markers of intrinsic apoptosis, in each muscle tissue at 12 weeks after cell administration compared to the control and Reference Example mice.

[0140] It is known that patients with sarcopenia have increased apoptosis of muscle cells. The cell preparation of the present invention can, for example, inhibit apoptosis of muscle cells, thereby inhibiting a decrease in muscle mass, and thus has been found to have a therapeutic effect on sarcopenia. Furthermore, since muscle mass is also reduced in age-related sarcopenia such as frailty due to apoptosis of muscle cells, it can be said that the cell preparation of the present invention also has a therapeutic effect on other age-related sarcopenias.

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

[0142] The gastrocnemius and soleus muscles of each mouse collected in Example 2 were analyzed by RT-PCR using the qPCR kit (Powers SYBR® Green CR Master Mix, ThermoFisher, Cat. No. #437659) to determine the mRNA expression levels of tumor necrosis factor-α (TNF-α), an inflammatory cytokine, and monocyte chemotactic protein-1 (MCP-1), a chemokine. Furthermore, each collected muscle tissue was stained for CD68 to detect macrophages. The results are shown in Figure 10. Primer set for TNF-α 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 showing the expression levels of inflammatory cytokines and photographs of muscle tissue. Figure 10(A) shows graphs showing 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 in the gastrocnemius muscle, and the bottom two graphs show the results in the soleus muscle. The top and bottom graphs of Figure 10(A) show, from left to right, the results for TNF-α and MCP-1. In each graph of Figure 10(A), the vertical axis shows the relative expression level of TNF-α or MCP-1 mRNA relative to the endogenous control (GAPDH), and the horizontal axis shows the type of mouse. Figure 10(B) shows photographs showing the results of CD68 staining. The left four graphs show the results for the gastrocnemius muscle, and the right four graphs show 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] 10, the expression levels of TNF-α or MCP-1 mRNA in the gastrocnemius and soleus muscles of the mice of Examples A and B were reduced compared to the control mice 12 weeks after cell administration. Furthermore, the results of CD68 staining also revealed that the amount of macrophages in the gastrocnemius and soleus muscles of the mice of Examples A and B was reduced compared to the control mice.

[0145] In the mice of Examples A and B, the expression levels of TNF-α and MCP-1 in muscle tissue were reduced, and the amount of macrophages in muscle tissue was reduced, demonstrating that the cell preparation of the present invention can suppress inflammation in muscle tissue. As aging progresses, it is said that minute inflammation occurs in muscle tissue, which causes 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 exerting a therapeutic effect on sarcopenia.

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

[0147] The gastrocnemius and soleus muscles of each mouse collected in Example 2 were analyzed by RT-PCR to confirm the expression level of TGF-β1 mRNA in the muscle tissue using the qPCR kit (Powers SYBR® Green CR Master Mix, ThermoFisher, Cat. No. #437659). Each collected muscle tissue was stained for PCNA (proliferating cell nuclear antigen) to detect the proliferating cell nuclear antigen in each muscle tissue, and Desmin+ / Laminin5+ fluorescent double staining was performed to confirm the expression of desmin and laminin 5 in each muscle tissue. 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 a graph showing the expression level of TGF-β1 and photographs of muscle tissue. In Figure 11, (A) is a graph showing the expression level of TGF-β1 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 graph in Figure 11(A), the vertical axis shows the relative expression level of TGF-β1 mRNA to the endogenous control (GAPDH), and the horizontal axis shows the type of mouse. In Figure 11(B), the four graphs on the left show the results in the gastrocnemius muscle, and the four graphs on the right show the results in the soleus muscle. In the four left and right figures 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), the area of ​​proliferating cell nuclear antigen stained by PCNA staining is indicated by a white triangle (▽). Figure 11(C) is a photograph showing the results of Desmin / Laminin5 fluorescent double staining, with the four left figures showing the results for the gastrocnemius muscle and the four right figures showing the results for the soleus muscle. In the four left and right figures 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), the Desmin stained by Desmin / Laminin5 fluorescent double staining is indicated by a white triangle (▽). + / Laminin5 + Double positives are indicated with a white asterisk (*).

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

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

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

[0152] The gastrocnemius and soleus muscles of each mouse collected in Example 2 were analyzed by RT-PCR using the qPCR kit (Powers SYBR® Green CR Master Mix, ThermoFisher, Cat. No. #437659) to determine the expression levels of mRNA encoding cathepsin K (CatK) and GAPDH. Furthermore, the expression levels of Sirt1 and myosin heavy chain (MHC), as well as GAPDH, were determined 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 and photographs showing 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 levels of Sirt1 in the gastrocnemius, and (C) is a gel image and graph showing the expression levels of MHC in the gastrocnemius. In Figure 12(A), the left image is a graph showing the results in the gastrocnemius, and the right image is a graph showing the results in the soleus. In each graph in Figure 12(A), the vertical axis indicates the relative expression level of CatK relative to the endogenous control (GAPDH), and the horizontal axis indicates the type of mouse. In the gel images of Figures 12(B) and (C), the upper row shows the expression levels of Sirt1 or MHC, and the lower row shows the expression level of the endogenous control (GAPDH). In each gel image, the third lane from the left shows the results for the control (Control), Example A (Cell), Reference Example (Ex), and Example B (Cell+Ex). In the graphs of Figures 12(B) and (C), the vertical axis indicates the relative expression level of Sirt1 or MHC relative to the endogenous control (GAPDH), and the horizontal axis indicates the type of mouse.

[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 12 weeks after cell administration compared to the control mice. Furthermore, the mice of Examples A and B showed a significant increase in Sirt1 and MHC expression 12 weeks after cell administration compared to the control and reference mice.

[0155] CatK is known to be highly expressed after muscle damage, and by inducing inflammation, it enhances muscle damage and delays regeneration. Sirtuin genes, such as Sirt1, are also known to be genes associated with anti-aging effects. MHC is a gene associated with the repair and increase of muscle mass and is known to function in maintaining muscle fiber mass. Therefore, the cell preparation of the present invention can suppress the expression level of CatK in skeletal muscle and increase the expression levels of Sirt1 and MHC, and therefore it is presumed that it can enhance and maintain skeletal muscle repair. Therefore, it has been found that the cell preparation of the present invention suppresses the expression of genes that enhance muscle damage, while inducing the expression of genes that regenerate muscle, thereby suppressing the loss of muscle mass and thereby exhibiting a therapeutic effect on sarcopenia.

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

[0157] (1) Purification of exosomes from cell culture supernatant To confirm that the exosomes in the cell preparation of the present invention inhibit apoptosis, exosomes were first purified from the cell preparation culture supernatant. Specifically, the cell preparation prepared in the same manner as in Example 1(1) to (3) was placed on a Corning® CellBIND® surface 100 mm dish (manufactured by Corning Incorporated, Product Number: #3292) at a cell count of 1 x 10 cells. 5The cells were seeded at a density of 1000 cells / 1 ml and cultured for 48 hours. The culture medium used for the culture was CiMS™-BM (Nipro Corporation, product code: 87-070) supplemented with CiMS™-sAF (Nipro Corporation, product code: 87-072), an animal-free additive for basal culture medium for human mesenchymal stem cells, at a ratio according to the package insert. After the culture, the cells were centrifuged at 300 × g for 10 minutes to remove the cells and obtain a first supernatant. The supernatant was centrifuged at 2000 × g for 10 minutes to remove cell fragments and obtain a second supernatant. The second supernatant was then filtered using a 0.22 μm filter to obtain a sample. The sample was placed in an ultracentrifuge UC tube and placed in a rotor (SW32Ti-12U, Beckman Coulter). A third centrifugation was performed using an ultracentrifuge (Optima L-100, Beckman Coulter). The third centrifugation was performed at 35,000 rpm for 70 minutes. After the third centrifugation, the supernatant was removed to obtain a precipitate. 1 ml of filtered PBS was added to the precipitate and vortexed. After vortexing, the volume was adjusted to 35 ml with PBS, and a fourth centrifugation was performed under the same conditions as the third centrifugation. After the fourth centrifugation, the supernatant was removed, a small amount of PBS was added, and the mixture was vortexed to obtain an exosome solution. The exosome solution was then stored in a 1.5 ml low-binding 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 allowed to air dry 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, manufactured by JEOL Ltd.). These results are shown in Figure 13.

[0159] Figure 13 is an electron micrograph of exosomes. In Figure 13, the scale bar indicates 200 nm. As shown in Figure 13, numerous round vesicles with diameters ranging from approximately 50 mm to approximately 200 mm were observed. These findings suggest that exosomes can be purified from the culture supernatant of the cell preparation of the present invention.

[0160] (3) Exosome marker confirmation To confirm whether the round vesicles observed under the electron microscope were exosomes, Western blotting was used to confirm whether the vesicles expressed exosome markers. Specifically, 50 μl of 2x sample buffer was added to 50 μl of the exosome solution obtained in Example 8(1) above, and the mixture was heated at 95°C for 5 minutes to prepare a sample. After preparation, the sample was applied to the wells of a 10-20% SuperSep™ Ace (Fujifilm Wako Pure Chemical Industries, Ltd.) acrylamide gel and subjected to electrophoresis. After electrophoresis, Western blotting was performed. For exosome detection in Western blotting, the sample was incubated with a primary antibody overnight, followed by staining with a secondary antibody at room temperature (approximately 25°C) for 1 hour. The primary antibodies used were exosome markers: 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 for three additional exosome solutions prepared independently, and the results are shown in Figure 14.

[0161] Figure 14 is a photograph showing the results of exosome marker detection by Western blotting. In Figure 14, the left side of the photograph shows the exosome marker, the upper part shows the exosome solution sample, and lanes 1 to 4 each show the results of an independently prepared exosome solution. As shown in Figure 14, exosome markers were detected in all exosome solution samples, reproducibly confirming that umbilical cord-derived mesenchymal stem cells secrete exosomes. From the above, it was demonstrated that exosomes can be purified from the culture supernatant of the cell preparation of the present invention.

[0162] (4) Evaluation of exosome particle size distribution 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 dilution, the particle size of the exosomes was measured using a particle size analyzer (Nano particle tracking analysis (NanoSight), manufactured by Malvern Panalytical) with default parameters. The results are shown in Figure 15.

[0163] Figure 15 is a graph showing the particle size distribution of exosome markers. In Figure 15, the vertical axis represents exosome concentration (number of vesicles / ml), and the horizontal axis represents exosome size (nm). As shown in Figure 15, exosomes from the umbilical cord-derived mesenchymal cells were distributed in the 40-400 nm range, with the most abundant exosomes being 116 nm, the smallest being 40 nm, and the largest being 621 nm. Furthermore, exosomes ranging in size from approximately 100 nm to approximately 300 nm were frequently observed. From these findings, it was determined that the exosomes obtained from the culture supernatant of the cell preparation of the present invention were exosomes ranging in size from 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 incorporated into cultured muscle cells. Specifically, the exosome solution (10 μg protein amount) obtained in Example 8(1) was labeled using ExoSparkler Exosome Membrane Labeling Kit-Green (Cat. No.: EX01, Dojindo Laboratories). After labeling, 1.25 × 10 exosomes were placed in a dish. 4 The cells were added to seeded C2C12 cells (mouse striated muscle cells). After the addition, the cells were incubated for 24 hours. After the incubation, the cells were fixed at room temperature for 20 minutes using 4% paraformaldehyde-containing phosphate buffer (Cat. No. 161-20141, Fujifilm Wako Pure Chemical Industries, Ltd.). After the fixation, the cells were washed with PBS at room temperature for 5 minutes, and this washing was repeated three times in total. The cells were then observed using a fluorescence microscope (Super-resolution / confocal Microscopy (LSM880-ELYRA PS. 1, Carl Zeiss). The 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 C2C12 cells have taken 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 inhibit apoptosis in cultured muscle cells that had taken up the exosomes. Specifically, 2 × 10 C2C12 cells (mouse striated muscle cells) were cultured in a 12-well culture plate containing a cover glass. 4After seeding the cells, 300 μmol / L of H2O2 was added. After the addition, the cells were incubated for 12 hours. After the incubation, the cells were cultured in DMEM medium to which the exosome solution (1 μg in terms of protein amount) obtained in Example 8(1) was added. As a negative control, the cells were cultured using DMEM medium to which an equal volume of PBS was added instead of the exosome solution. The culture was continued for 12 hours. After the culture, TUNEL staining was performed, and the cells were observed using a fluorescence microscope. In addition, ProLong TM Galss Antifade Mountan with NucBlue TM (Cat. No. P36985, ThermoFisher) was used. The proportion of TUNEL-positive cells among nuclear-positive cells was calculated. The results are shown in Figure 17. T tests (and nonparametric tests) were used to test for significance.

[0167] Figure 17 shows photographs and graphs illustrating apoptosis in C2C12 cells treated with 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 indicates the presence or absence of exosome addition, and the top of the photograph indicates the type of marker. In Figure 17(B), the vertical axis indicates the percentage of TUNEL-positive cells, and the horizontal axis indicates the presence or absence of exosome addition. As shown in Figure 17(A), fewer TUNEL-positive cells were observed in C2C12 cells treated with exosomes (exosomes) compared to the negative control (control) without exosome addition. Furthermore, as shown in Figure 17(B), the percentage of TUNEL-positive cells was significantly reduced in C2C12 cells treated with exosomes compared to the negative control 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 induced by H2O2 in muscle cells that have taken up the exosomes. Furthermore, the present inventors are the first to demonstrate that umbilical cord-derived cells secrete exosomes.

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

[0169] This application claims priority based on Japanese Patent Application No. 2020-190551, filed on November 16, 2020, the disclosure of which is incorporated herein in its entirety.

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

Claims

1. A cell preparation for use in suppressing age-related muscle mass loss, comprising: The cell preparation comprises: The cells are derived from umbilical cords and grown in a culture medium containing a basal culture medium for human mesenchymal stem cells. The umbilical cord-derived cells (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2; and (ii) negative for CD45, CD34, CD11b, CD19, and HLA-Class II; The umbilical cord-derived cells have an effect of inducing the expression of SIRT1 gene and / or MHC gene in muscle tissue. Cell preparation.

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

3. The umbilical cord-derived cells (iii) The cell preparation according to claim 1 or 2, wherein expression of any one of the gene and / or protein of IDO, PGE2, and PD-L1 is induced under inflammatory conditions.

4. The cell preparation of claim 1 , wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue and / or Wharton's jelly.

5. The cell preparation was 1 x 10 ６ ~1 x 10 ９ 5. The cell preparation of claim 1 , comprising umbilical cord-derived cells.

6. The cell preparation of claim 1 , wherein the cell preparation comprises an extract and / or secretion of the umbilical cord-derived cells.

7. The cell preparation of claim 1 , wherein the cell preparation exhibits an effect of improving mitochondrial function in muscle tissue.

8. The cell preparation of claim 1 , wherein the cell preparation exhibits the effect of increasing the number of mitochondria in muscle tissue.

9. The cell preparation according to claim 1 , wherein the cell preparation exhibits an effect of inducing the expression of a gene that improves mitochondrial function in muscle tissue.

10. The cell preparation according to claim 9, wherein the mitochondrial function-improving gene is the PGC1-α gene, the COX4 gene, and / or the GLUT4 gene.

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

12. The cell preparation of claim 1 , wherein the cell preparation exhibits anti-inflammatory activity in muscle tissue.

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

14. The cell preparation of claim 13, wherein the inflammatory cytokine gene is a TNF-α gene.

15. The cell preparation of claim 13, wherein the chemokine gene is the MCP-1 gene.

16. The cell preparation according to claim 1 , wherein the cell preparation exhibits a proliferation-promoting activity on muscle cells.

17. The cell preparation according to claim 1 , wherein the cell preparation exhibits an activity of inducing expression of a gene that promotes proliferation of muscle cells.

18. The cell preparation of claim 17, wherein the muscle cell proliferation-promoting gene is the TGF-β1 gene.

19. The cell preparation according to any one of claims 1 to 18, wherein the cell preparation exhibits a muscle tissue repair effect.

20. The cell preparation according to any one of claims 1 to 19, wherein the cell preparation exhibits an inhibitory effect on muscle tissue damage.

21. The cell preparation according to claim 1 , wherein the cell preparation exhibits an inhibitory effect on the expression of muscle tissue injury-inducing genes.

22. The cell preparation of claim 21 , wherein the muscle tissue damage-inducing gene is a cathepsin K gene.

23. A cell preparation according to any one of claims 1 to 22 for use in conjunction with exercise therapy.

24. A cell preparation according to any one of claims 1 to 23 for use in a subject suffering from inflammation in muscle tissue.

25. 25. The cell preparation of any one of claims 1 to 24, for intravenous administration.

26. The cell preparation of claim 1 , wherein the loss of muscle mass is due to aging.

27. A cell preparation according to any one of claims 1 to 26, for administration to middle-aged and elderly people.

28. A cell preparation for use in the treatment of age-related sarcopenia, comprising a cell preparation for use in inhibiting loss of muscle mass described in any one of claims 1 to 27.

29. The cell preparation of claim 28, wherein the age-related muscle loss is sarcopenia or frailty.

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