Extracellular secretion and use therefor

Abstract

The present disclosure provides an extracellular secretion that is useful in the treatment of various diseases. In one embodiment, the present disclosure provides an extracellular secretion including approximately 6×1010 / mL or more small extracellular vesicles (sEVs). In one embodiment, the present disclosure provides an extracellular secretion including small extracellular vesicles (sEVs) having a particle size distribution in which the particle size mode is approximately 100 nm to approximately 120 nm. In one embodiment, the extracellular secretion is obtained from a culture supernatant of mesenchymal stem cells. In one embodiment, the extracellular secretion is used to treat aging.

Description

Extracellular secretions and their use

[0001] This disclosure relates to extracellular secretions and their uses.

[0002] Cell-based medical treatments, such as regenerative medicine using iPS cells and CAR-T therapy, are being developed. However, cell-based therapies have problems, such as the risk of cancer.

[0003] The inventors of this invention have discovered an extracellular secretion useful for treating diseases, and have completed the present invention.

[0004] This disclosure provides, for example, the following items: (Item 1) Approximately 6 x 10 １０(Item 2) Extracellular secretions containing small extracellular vesicles (sEVs) of a certain size per mL or more. (Item 3) Extracellular secretions containing extracellular vesicles (EVs) having a particle size distribution with a mode of approximately 100 to approximately 120 nm. (Item 4) Extracellular secretions according to item 1 or 2, wherein the proportion of small extracellular vesicles (sEVs) among all extracellular vesicles is approximately 90% or more. (Item 4) Extracellular secretions according to item 1 or 2, obtained from the culture supernatant of mesenchymal stem cells. (Item 5) Extracellular secretions according to item 4, obtained from the culture supernatant of mesenchymal stem cells cultured in a medium containing sodium bicarbonate and having a pH of approximately 8.0 to approximately 8.5. (Item 6) Compositions comprising extracellular secretions for treating or preventing aging or age-related diseases. (Item 7) Compositions according to item 6, wherein the age-related disease includes Alzheimer's disease or Parkinson's disease. (Item 8) The composition according to Item 6, wherein the aging-related disease includes diabetes mellitus. (Item 9) The composition according to Item 6, wherein the aging-related disease includes cardiovascular disease. (Item 10) The composition according to Item 6, wherein the aging-related disease includes chronic obstructive pulmonary disease (COPD). (Item 11) The composition according to Item 6, wherein the aging-related disease includes arthritis and bone disease. (Item 12) A composition for regulating mTOR activity or MAPK activity, comprising an extracellular secretion. (Item 13) The composition according to any one of Items 6 to 12, wherein the extracellular secretion is obtained from the culture supernatant of mesenchymal stem cells. (Item 14) The composition according to any one of Items 6 to 12, wherein the extracellular secretion is the extracellular secretion according to any one of Items 1 to 5. (Item 15) An extracellular secretion according to any one of Items 1 to 5 or a composition according to any one of Items 6 to 14, for injection. (Item 16) A method for preparing a supernatant fraction or extracellular vesicle fraction of an extracellular secretion, comprising the step of separating the extracellular secretion described in any one of Items 1 to 5 into a supernatant fraction and an extracellular vesicle fraction. (Item 17) A supernatant fraction, an extracellular vesicle fraction, or a combination comprising the supernatant fraction and the extracellular vesicle fraction obtained by the method described in Item 16.(Item 18) A method for testing extracellular secretions, comprising the steps of: incubating human cultured cells in a medium containing a predetermined amount of the extracellular secretions; and measuring the phosphorylation of one or more of mTOR, S6K1, ULK1, 4E-BP1, and ERK1 / 2 in cells incubated with the extracellular secretions, wherein the extracellular secretions are deemed acceptable if the phosphorylation is elevated compared to a control condition without extracellular secretions and is lower compared to the extracellular secretions from which EVs have been removed. (Item 19) A method for testing extracellular secretions, comprising the steps of: incubating human cultured senescent cells in a medium containing a predetermined amount of the extracellular secretions; and measuring one or more of β-Gal activity, cell proliferation activity, cell doubling time, mitochondrial activity, and DNA damage in cells incubated with the extracellular secretions, wherein the measured values ​​for the items are improved in terms of indicators of senescent cells compared to a control condition without extracellular secretions.

[0005] The extracellular secretions described herein are useful for treating various diseases.

[0006] This section outlines the procedure for isolating small-sized extracellular viable (EV) fractions (sEVs) from extracellular secretions. The particle size distribution of all EVs contained in the extracellular secretions is shown with error bars. The left panel shows the results of Western blots for phosphorylated mTOR and GAPDH under various sample conditions under Torin treatment (Torin-2: 250 nM for 8 hours). The right panel shows the quantitative results of phosphorylated mTOR under various sample conditions (shown as relative intensity to GAPDH). The numbers indicate the following sample conditions: 1: Untreated (Control), 2: ES (extracellular secretions), 3: Small-sized EV fraction (sEVs), 4: ES-S (centrifugation supernatant). p70 shows the results of Western blots for phosphorylation of S6 kinase (S6K). The left image shows the results of Western blots for S6K protein expression and phosphorylation under various sample conditions under Torin treatment (Torin-2: 250 nM for 8 hours). The right image shows the quantitative results of phosphorylated S6K under various sample conditions (shown as relative intensity to GAPDH). The numbers indicate the following sample conditions: 1: Untreated (Control), 2: ES (Extracellular secretion), 3: Small-size EV fraction (sEVs), 4: ES-S (Centrifugal supernatant). The left image shows the results of Western blots for phosphorylation of ULK1 (left) and 4E-BP1 (right) under various sample conditions under Torin treatment (Torin-2: 250 nM for 8 hours). The numbers indicate the following sample conditions: 1: Untreated (Control), 2: ES (Extracellular secretion), 3: Small-size EV fraction (sEVs), 4: ES-S (Centrifugal supernatant). In 4E-BP1, a band appears at the top as phosphorylation increases. Western blot results for ERK1 / 2 phosphorylation are shown. The numbers indicate the following sample conditions: 1: Untreated (Control), 2: ES (Extracellular secretion), 3: Small-size EV fraction (sEVs), 4: ES-S (Centrifugal supernatant). Western blot results for S6K protein expression and phosphorylation in each EXO-Prep treated sample with BafA1 (100 nM) are shown. BafA1 inhibits lysosome acidification, thereby suppressing mTOR activation. The right shows the quantitative results of S6K and phosphorylated S6K under each sample condition (shown as relative intensity to GAPDH).The numbers indicate the following sample conditions: 1: Untreated (Control), 2: BafA1 only, 3: BafA1 + ES (extracellular secretions), 4: EXO-Prep extracted EVs. A) Microscopic images of the LC3 staining tendency of cells cultured with or without extracellular secretions (ES) added along with BafA1. Puncta formation is observed under the condition with ES added. B) The number of cells and total number of cells showing puncta formation under the culture conditions with or without extracellular secretions (ES) added. C) Western blot results showing p62 and LC3 expression under culture conditions with or without BafA1 and extracellular secretions (ES) added. Mitotracker Red CM-H of cells cultured under each culture condition. ２ The results of staining with ROS are shown. When extracellular secretions are added (center), compared to the control condition, Mitotracker Red CM-H ２The fluorescence induced by ROS was greatly increased. Western blot results for S6K phosphorylation under culture conditions with and without the addition of extracellular secretions (ES) in the presence of tunicamycin at various concentrations are shown. The left is the Western blot image, and the right is the quantitative result. Changes in β-gal activity accompanying subculturing of established cells are shown. The vertical axis shows β-gal intensity, and the horizontal axis shows the number of subculturings. * indicates a significant difference of p < 0.05. A comparison of sEVs obtained from extracellular secretions in Example 1 and the comparative product (OC-01) is shown. The vertical axis shows the relative particle size distribution with error bars. The sEVs derived from extracellular secretions in Example 1 contain more particles. The effect of extracellular secretions on β-gal activity in senescent cells is shown. The vertical axis shows β-gal intensity, and the horizontal axis from left to right shows: untreated (Control), comparative product, 5% ES (extracellular secretions), 10% ES. * indicates a statistically significant difference of p < 0.05. This shows the effect of extracellular secretions on the proliferation of senescent cells. The vertical axis shows the relative value of WST-8 activity compared to untreated cells, and the horizontal axis shows, from left to right, the following: untreated (Control), comparison product, ES (extracellular secretions) 5%, ES 10%. * indicates a statistically significant difference of p < 0.05. This shows the effect of extracellular secretions on the doubling time of senescent cells. The vertical axis shows the doubling time (hours), and the horizontal axis shows, from left to right, the following: untreated (Control), NMN (nicotinamide mononucleotide) (1 mM), comparison product, ES (extracellular secretions) 5%, ES 10%. This shows the effect of extracellular secretions on mitochondrial activity (ATP) of senescent cells. The vertical axis shows the relative value of WST-8 activity compared to untreated cells, and the horizontal axis shows, from left to right: untreated (Control), ALA (5-aminolevulinic acid; 1 mM), comparison product, ES (extracellular secretion) 5%, ES 10%. * indicates a statistically significant difference of p < 0.05. This shows the effect of extracellular secretions on mitochondrial activity (NAD+) in senescent cells. The vertical axis shows the relative amount of NAD+ compared to untreated cells, and the horizontal axis shows, from left to right: untreated (Control), NMN (1 mM), comparison product, ES (extracellular secretion) 5%. This shows the relative percentage of γH2AX-positive cells in young and senescent cells. These are micrographs showing γH2AX expression when young and senescent cells are treated with extracellular secretions (ES) or Dox (doxorubicin).This shows the number of γH2AX staining spots when young and aged cells were treated with extracellular secretions (ES) or Dox (doxorubicin).

[0007] The present invention will be described below with reference to illustrative examples, as necessary, with reference to the accompanying drawings. Throughout this specification, singular expressions should be understood to include the concept of their plural forms unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0008] (Definition) In this specification, “extracellular secretions” (which may be abbreviated as “ES”) refer to substances produced by cells that are released outside the cell, or compositions containing such substances. Typically, extracellular secretions are the supernatant of a cell culture or its components (extracellular vesicles, proteins, etc.) that do not contain cells themselves. Extracellular secretions are also obtained by removing certain components (extracellular vesicles, proteins, etc.) from the supernatant of a cell culture.

[0009] In this specification, “extracellular vesicles” (sometimes abbreviated as “EVs”) refer to vesicles smaller than cells that are located outside the cell and surrounded by a lipid membrane. Extracellular vesicles typically contain proteins (e.g., membrane proteins), nucleic acids, and lipids. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles typically have a diameter of about 50 to 5000 nm. In this specification, sEVs refer to extracellular vesicles with a particle size of about 50 to 600 nm. The particle size of extracellular vesicles can be measured, for example, by nanotracking.

[0010] In this specification, “mesenchymal stem cells” are cells that have at least one of the following characteristics: (1) expression of specific cell membrane markers CD73, CD90, and CD105; (2) lack of expression of CD11b, CD14, CD34, CD45, CD19, CD79a, and HLA-DR; and (3) three germ layer pluripotency (the ability to differentiate into osteoblasts, chondrocytes, and adipocytes). Mesenchymal stem cells can be obtained from bone marrow, adipose tissue, teeth, etc., and can also be differentiated from pluripotent cells.

[0011] In this specification, the term "approximately" refers to plus or minus 10% of the indicated value unless otherwise specified. When "approximately" is used for temperature, it refers to plus or minus 5°C of the indicated temperature; when "approximately" is used for pH, it refers to plus or minus 0.5 of the indicated pH.

[0012] (Preferred Embodiments) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure is not limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure by taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination.

[0013] (Extracellular secretions) In one aspect, the present disclosure provides extracellular secretions. In one embodiment, the extracellular secretions include the culture supernatant or components of mesenchymal stem cells. Therefore, the extracellular secretions may include components of the culture medium used to culture the cells (e.g., salts, buffers, growth factors, vitamins, minerals, cytokines, etc.). In one embodiment, the extracellular secretions do not contain cells. Cells can be easily removed from the culture supernatant using a filter or the like.

[0014] Typically, the extracellular secretions of the present disclosure are those with adjusted concentrations of the cell culture supernatant or its extracellular vesicle components. As shown in the examples, in the culture supernatant of mesenchymal stem cells, it was found that extracellular vesicles and other components have different functions in mTOR activity and MAPK activity. Therefore, by adjusting the concentration of the extracellular vesicle components in the culture supernatant, the mTOR activity and MAPK activity of the extracellular secretions can be regulated. Methods for adjusting the concentration of extracellular vesicle components include centrifugation, sucrose density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, etc. In particular, centrifugation can concentrate or reduce extracellular vesicles with a minimal impact on biological materials such as proteins contained in extracellular secretions.

[0015] In one embodiment, the extracellular secretion is characterized by small-sized extracellular vesicles (sEVs). Excessive mTOR activation can be suppressed by small-sized extracellular vesicles (sEVs). In one embodiment, the extracellular secretion contains small-sized extracellular vesicles (sEVs) at a concentration of about 1×10 １０ cells / mL or more, about 2×10 １０ cells / mL or more, about 5×10 １０ cells / mL or more, about 5×10 １０ cells / mL or more, about 1×10 １１ cells / mL or more, or about 5×10 １１ cells / mL or more, and / or about 5×10 １０ cells / mL or less, about 1×10 １１ cells / mL or less, about 2×10 １１ cells / mL or less, about 5×10 １１ cells / mL or less, or about 1×10 １２ cells / mL or less.

[0016] In one embodiment, the small-sized extracellular vesicles (sEVs) in the extracellular secretion have a particle size mode value of about 90 to about 130 nm, about 90 to about 120 nm, about 90 to about 115 nm, about 90 to about 110 nm, about 100 to about 130 nm, about 100 to about 120 nm, about 100 to about 115 nm, about 100 to about 110 nm, about 105 to about 130 nm, about 105 to about 120 nm, or about 105 to about 115 nm.

[0017] In one embodiment, the small extracellular vesicles (sEVs) in the extracellular secretion have a particle size distribution with a full width at half maximum of approximately 40–90 nm, 50–80 nm, 55–75 nm, or 60–70 nm.

[0018] In one embodiment, the extracellular vesicles in the extracellular secretion of the present invention may have a proportion of small-sized extracellular vesicles (sEVs) of about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the total extracellular vesicles.

[0019] In a typical embodiment, sEVs as used herein can be prepared by obtaining extracellular vesicles contained in a pellet obtained by centrifugating a material containing extracellular vesicles (such as the supernatant of a cell culture) at 10,000 G at 4°C for 40 minutes, and then centrifugating the supernatant at 100,000 G at 4°C for 70 minutes.

[0020] (Preparation of extracellular secretions) In one embodiment, extracellular secretions may be prepared from the culture supernatant of mesenchymal stem cells. In one embodiment, the mesenchymal stem cells are human cells. In one embodiment, the mesenchymal stem cells are derived from adipocytes, nerve cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, osteocytes, chondrocytes, germline cells, or iPS cells, and are preferably derived from adipocytes.

[0021] Cell culture conditions can affect the components of extracellular secretions (such as extracellular vesicles). In one embodiment, the extracellular secretions of the present disclosure are prepared from the culture supernatant of mesenchymal stem cells cultured at pH levels of approximately 7.0–10.0, 7.0–9.5, 7.0–9.0, 7.0–8.5, 7.5–10.0, 7.5–9.5, 7.5–8.5, 8.0–10.0, 8.0–9.5, 8.0–9.0, 8.0–8.5, 8.5–10.0, or 8.5–9.5. In one embodiment, the culture medium for mesenchymal stem cells comprises Tris hydrochloride buffer, HEPES buffer, or sodium bicarbonate buffer, preferably sodium bicarbonate buffer. In one embodiment, the culture medium for mesenchymal stem cells contains magnesium (for example, about 0.001 mM to about 1 mM, e.g., about 0.001 mM, about 0.01 mM, about 0.1 mM, about 1 mM, etc.).

[0022] To prepare extracellular secretions from cell culture supernatant, operations such as integration and distribution of the collected culture supernatant, freezing, and filtering can be performed.

[0023] In one embodiment, the present disclosure provides a method for separating a cell culture supernatant into an extracellular vesicle fraction and a supernatant fraction. A fraction containing a higher concentration of extracellular vesicles compared to the original cell culture supernatant is referred to as the extracellular vesicle fraction, and a fraction containing a lower concentration of extracellular vesicles is referred to as the supernatant fraction. Methods such as centrifugation, sucrose density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, etc. can be used for separation. The present disclosure also provides an extracellular vesicle fraction and a supernatant fraction. In one embodiment, the extracellular vesicle fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at a concentration of at least about 110%, at least about 120%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 500%, at least about 700%, at least about 1000%, at least about 1500%, or at least about 2,000% of the cell culture supernatant. In one embodiment, the supernatant fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at a concentration of up to about 90%, up to about 80%, up to about 50%, up to about 20%, up to about 15%, up to about 10%, up to about 7%, up to about 5%, up to about 2%, up to about 1.5%, or up to about 1% of the cell culture supernatant. The present disclosure also provides a combination of the extracellular vesicle fraction and the supernatant fraction. Extracellular secretions that have not been separated into the extracellular vesicle fraction and the supernatant fraction may be beneficial by causing moderate activation of the mTOR pathway, but it may also be beneficial to use the extracellular vesicle fraction and the supernatant fraction separately in the regulation of the activation of the mTOR pathway or the MAPK pathway.

[0024] In one embodiment, the present disclosure provides a method for evaluating the mTOR pathway and / or MAPK pathway activation ability of extracellular secretions (e.g., cell culture supernatant). Since the performance of extracellular secretions can vary due to various factors in culture, a method for evaluating the performance of extracellular secretions may be useful to ensure the usefulness of extracellular secretions. In one embodiment, the extracellular secretions of the present disclosure may have a higher mTOR pathway and / or MAPK pathway activation ability when an operation to remove sEVs, such as centrifugation, is performed compared to when this removal operation is not performed.

[0025] (Composition) In one embodiment, the Disclosure provides a composition comprising the extracellular secretions of the Disclosure. The composition may include, for example, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericides, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, and the like.

[0026] The compositions described herein may be provided in various forms. These forms may include, for example, injectable preparations, reconstituted powders (e.g., lyophilized preparations), etc., but are preferably in solution form. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. The aqueous solutions for injection may also contain, for example, physiological saline, sugar (e.g., trehalose), NaCl, or NaOH. In preferred embodiments, the compositions can be formulated as pharmaceutical compositions adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. Generally, the components may be supplied separately or mixed together in unit dose forms, and can be supplied as lyophilized powders or water-free concentrates in sealed containers such as ampoules or sachets indicating the amount of the activator. When administering the composition by injection, it is also possible to dispense it using an injection bottle containing sterile-grade water or saline solution. When administering the composition by injection, it is also possible to provide an ampoule of sterile water or saline solution for injection so that the components can be mixed before administration. The composition may have a pH of, for example, about 5.0 to 9.0, about 5.5 to 8.5, or about 6.0 to 8.0.

[0027] The amount of the composition of the present disclosure may vary depending on the nature of the disorder or condition to be treated or prevented, but can be determined by standard clinical techniques based on the descriptions herein. In some cases, in vitro assays can also be used to assist in identifying the optimal dosage range. The exact dosage to be used in the formulation can also vary depending on the route of administration and the severity of the disease or condition, and thus should be determined according to the judgment of the attending physician and the circumstances of each patient. The dosing interval is not particularly limited, and for example, it may be administered once or twice per 1, 7, 14, 21, or 28 days, or once or twice per range of any two of these values. The dosage, dosing interval, and dosing method may be appropriately selected depending on the patient's age, weight, symptoms, etc.

[0028] The route of administration of the composition described herein may be, for example, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracerebral, epidural, etc. It may also be topical administration, but even for local diseases, they can be treated by intravenous administration.

[0029] The composition of the present disclosure can be provided as a kit. In one embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components that can be added to the composition of the present disclosure. In some cases, information indicating approval by a government agency for the manufacture, use, or sale for human administration in a form specified by the government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products may also be indicated in connection with such containers.

[0030] (Applicable Diseases) In one embodiment, the extracellular secretion or composition of the present disclosure is used for treating or preventing aging or for regulating mTOR (e.g., mTORC1) and / or MAPK. The present disclosure also provides methods of using the extracellular secretion or composition of the present disclosure as such. From the data shown in the examples, it was confirmed that the extracellular secretion of the present disclosure is particularly useful in the treatment or prevention of aging or aging-related diseases.

[0031] Aging can refer to both cellular aging and biological aging. Cellular aging is typically a state in which cells maintain a viable state and metabolic activity, but cell division has stopped and they have lost their proliferative capacity. While cellular aging is often observed in the cells of elderly individuals, it does not necessarily occur only in elderly individuals, and the application of the extracellular secretions or compositions of this disclosure is not limited to the elderly. Senescent cells are characterized by high aging-related β-galactosidase activity (SA-β-gal), high expression of p16 (cyclin-dependent kinase inhibitor 2A) or p21 (cyclin-dependent kinase inhibitor 1), accumulation of DNA damage markers (such as γ-H2AX), and decreased mitochondrial ATP production.

[0032] In one embodiment, the extracellular secretions or compositions of the present disclosure exhibit: • Higher aging-related β-galactosidase activity than normal cells (e.g., 1.1 times or more, 1.5 times or more, 2 times or more, etc.) (measurable by staining, etc.; see also Examples) • Lower proliferation than normal cells (e.g., 0.9 times or less, 0.7 times or less, 0.5 times or less, etc.) (measurable by WST-8 assay, etc.; see also Examples) • Higher p16 and / or p21 expression than normal cells (e.g., 1.1 times or more, 1.5 times or more, 2 times or more, etc.) (measurable by immunohistochemistry, PCR, etc.) • Higher accumulation of DNA damage markers than normal cells (e.g., 1.1 times or more, 1.5 times or more, 2 times or more, etc.) (measurable by immunohistochemistry, PCR, etc.) It is used to administer to subjects having a condition characterized by containing cells (e.g., cells from a lesion site) that have one or more of the following characteristics: ATP or NAD+ levels (which can be measured by chemiluminescence, etc.; see also Examples) that are lower than those of normal cells (e.g., 0.9 times or less, 0.7 times or less, 0.5 times or less, etc.). The normal cells used for comparison may be the same type of cells in a healthy person, the same type of cells in a healthy area of ​​the subject, etc.

[0033] In one embodiment, specific age-related diseases to be treated or prevented include Alzheimer's disease, Parkinson's disease, diabetes (especially type 2 diabetes), cardiovascular diseases (such as arteriosclerosis, hypertension, myocardial infarction, and stroke), chronic obstructive pulmonary disease (COPD), arthritis, and bone diseases (such as osteoarthritis and osteoporosis). These diseases can be treated or prevented by modulation (activation) of the mTOR pathway and / or the MAPK pathway. Furthermore, it is expected that the extracellular secretions or compositions of this disclosure can also treat or prevent aging of organisms composed of cells (e.g., the human body) by treating or preventing cellular aging.

[0034] (Examination of Extracellular Secretions) In one embodiment, the present disclosure provides a method for examining extracellular secretions and a method for selecting extracellular secretions based on the results. The examination may be based on one or more of the EV composition, effects on the mTOR pathway and / or the MAPK pathway, and effects on cellular senescence.

[0035] In one embodiment, the test may include determining the EV composition contained in extracellular secretions. The test is: 1 × 10 １０ pcs / mL or more, 2 x 10 １０ More than 10 cells / mL or 6 x 10 １０ Total EVs of 1 x 10¹ / mL or more, １０ pcs / mL or more, 2 x 10 １０ More than 10 cells / mL or 6 x 10 １０ Extracellular secretions that satisfy one or more of the following conditions can be judged as acceptable: sEVs of 1 / mL or more, a mode of particle size of 95-125 nm or 100-120 nm, and a full width at half maximum of 55-75 nm or 60-70 nm.

[0036] In one embodiment, the test may include determining the effect of extracellular secretions on the mTOR pathway and / or the MAPK pathway. The test involves incubating human cultured cells (e.g., human neuroblastoma-derived cells) for a predetermined time (e.g., 8 hours) in a medium containing a predetermined amount (e.g., 5 vol%) of test extracellular secretions (and optionally 250 nmol / L of Torin-2) and determining that phosphorylation of one or more of mTOR, S6K1, ULK1, and 4E-BP1 is elevated compared to a control condition without extracellular secretions (e.g., an increase of 1.1 times or more, 1.5 times or more, or 2 times or more in the amount of phosphorylated protein quantified), and is decreased compared to test extracellular secretions from which EVs have been removed (e.g., by centrifugation) (e.g., an increase of 0.9 times or less, 0.7 times or less, or 0. Extracellular secretions can be deemed acceptable if they satisfy one or more of the following conditions: a decrease of five times or less, and when human cultured cells (e.g., human neuroblastoma-derived cells) are incubated for a predetermined time (e.g., 8 hours) in a medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion, the phosphorylation of ERK1 / 2 is increased compared to a control condition without extracellular secretions (e.g., an increase of 1.1 times or more, 1.5 times or more, or 2 times or more in the amount of phosphorylated protein quantified), and is decreased compared to the test extracellular secretion from which EVs have been removed (e.g., by centrifugation) (e.g., a decrease of 0.9 times or less, 0.7 times or less, or 0.5 times or less in the amount of phosphorylated protein quantified). Quantification of phosphorylated proteins can be performed by measuring luminescence intensity by immunostaining, ELISA, etc. Control conditions (conditions without extracellular secretions, conditions with EVs removed, etc.) may be replaced with specified values ​​(specified values ​​can be set by repeated measurements under the same test conditions, etc.).

[0037] In one embodiment, the test may include determining the effect on cellular senescence.The test involves: • Incubating human cultured senescent cells (e.g., human dermal fibroblasts) in a medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion for a predetermined time (e.g., 72 hours), and confirming that β-Gal activity (e.g., fluorescence intensity measured using a β-Gal measurement kit) decreases compared to a control condition without the extracellular secretion (e.g., a decrease of 0.9 times or less, 0.7 times or less, or 0.5 times or less in the quantified fluorescence intensity); • Incubating human cultured senescent cells (e.g., human dermal fibroblasts) in a medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion for a predetermined time (e.g., 48 hours), and confirming that cell proliferation activity (which can be evaluated by WST-8 assay, etc.) increases compared to a control condition without the extracellular secretion (e.g., an increase of 1.1 times or more, 1.5 times or more, or 2 times or more in the quantified fluorescence intensity); - When human cultured senescent cells (e.g., human dermal fibroblasts) are incubated for a predetermined time (e.g., 48 hours) in a culture medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion, the cell doubling time (which can be calculated based on the number of cells after seeding and culturing a predetermined number of cells) is shortened (e.g., a reduction of 0.9 times or less, 0.7 times or less, or 0.5 times or less) compared to a control condition without the extracellular secretion. Extracellular secretions can be deemed acceptable if they satisfy one or more of the following conditions: • When human cultured senescent cells (e.g., human dermal fibroblasts) are incubated for a predetermined time (e.g., 48 hours) in a medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion, mitochondrial activity (which can be evaluated by ATP assays and NAD / NADH assays, etc.) increases compared to control conditions without the extracellular secretion (e.g., an increase of 1.1 times or more, 1.5 times or more, or 2 times or more in the quantified luminescence or fluorescence intensity); or • When human cultured senescent cells (e.g., human dermal fibroblasts) are incubated for a predetermined time (e.g., 48 hours) in a medium containing a predetermined amount (e.g., 5% by volume) of the test extracellular secretion, DNA damage (which can be evaluated by γH2AX expression levels, etc.) decreases compared to control conditions without the extracellular secretion (e.g., a decrease of 0.9 times or less, 0.7 times or less, or 0.5 times or less in the quantified fluorescence intensity).Human cultured senescent cells can be used if they have been confirmed to exhibit characteristics of senescent cells in terms of the indicators to be measured (β-Gal activity, proliferative capacity, etc.) compared to human cultured cells derived from the same type of tissue. Control conditions (such as conditions without extracellular secretions) may be replaced with specified values ​​(specified values ​​can be established by repeated measurements under the same test conditions).

[0038] In one embodiment, the above tests can be performed on the cell culture supernatant, and the extracellular secretions of this disclosure can be prepared using the culture supernatant that is deemed acceptable.

[0039] (Other Embodiments) The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0040] (Example 1: Extracellular vesicles (EVs) in extracellular secretions) Adipose-derived mesenchymal stem cells were thawed, and after centrifugation (800 rpm, 5 min), the supernatant was discarded. Then, fresh cell culture medium was added, and the cells were cultured. The pH was adjusted to approximately 8.0 with sodium bicarbonate, and the culture supernatant was obtained after removing the cells themselves and cell debris, and this was used as the extracellular secretion. As shown below, the extracellular secretion showed excellent effects, so the EVs contained therein were analyzed. In the following examples, unless otherwise specified, extracellular secretion (ES) refers to the entire culture supernatant from which extracellular vesicles have not been separated.

[0041] - Isolating extracellular vesicles (EVs) cultures were centrifuged at 4°C, 500G for 15 minutes to remove cell debris, etc., and the supernatant was used as extracellular secretions. 30 mL of the extracellular secretions were further centrifuged at 4°C, 10,000G for 40 minutes. The supernatant after the first centrifugation contained small-sized EVs, and the pellet contained medium- to large-sized EVs. The pellet containing medium- to large-sized EVs was resuspended in 1 mL of phosphate-buffered saline (PBS) and centrifuged again at 4°C, 10,000G for 40 minutes. The pellet after centrifugation was resuspended (600 μL) in phosphate-buffered saline (PBS) and this was used as the medium / large-sized EVs (m / l EVs). The supernatant after the first centrifugation was filtered through a 0.22 μm filter to remove impurities. The filtered supernatant was then centrifuged again at 4°C, 100,000G for 70 minutes. The pellet was suspended in PBS and subjected to a third centrifuge at 100,000 G for 70 minutes at 4°C. After the third centrifuge, the pellet was resuspended in PBS (600 μL). This was designated as small-size extracellular vesicles (sEVs). The supernatant after the second centrifuge was collected and designated as "ES-S". A Himac CP80NX with a P65A rotor was used for centrifugation at 100,000 G. The presence of extracellular vesicles in the extracellular secretions was confirmed by the use of CD63 and CD81 markers.

[0042] EV Size Measurement: The above sEVs were diluted 100 times (v / v) with PBS, and the EV size was measured using a NanoSight NS300 (Malvern, UK) CMOS camera, a violet laser (405 nm, <65 mW), and analysis software: NTA3.4. Measurements were performed five times.

[0043] The results are shown in Figure 2. The extracellular secretions contained approximately 6.15 × 10⁻⁶ １０ Total EVs per mL: approximately 6.1 × 10⁻⁶ １０ The sample contained sEVs (with particle sizes ranging from 50 to 600 nm) at a concentration of particles / mL, with a mode particle size of approximately 108 nm and a full width at half maximum of approximately 66 nm. The majority of the EVs were sEVs.

[0044] (Example 2: Relationship between extracellular secretions and EVs and mTOR and MAPK pathway activity) Based on the results of the study in this example, it was predicted that extracellular secretions have activity via the mTOR and MAPK pathways. It is known that activation of the mTOR pathway improves the phosphorylation of mTOR, as well as the phosphorylation of downstream molecules such as Atg13, ULK1, S6K1, and 4E-BP1, and that activation of the MAPK pathway improves the phosphorylation of ERK1 / 2. Therefore, the effects of extracellular secretions and EVs on these molecules were investigated. Typically, the experiment was conducted according to the following procedure.

[0045] Cell culture: SH-SY5Y (derived from human neuroblastoma) was cultured in Dulbecco's modified Eagle medium / Ham F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin with 5% CO2. ２ HeLa cells and 293T cells were cultured at 37°C in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin with 5% CO2. ２ The cells were cultured at 37°C. HDF (human fibroblasts) were cultured using Fibroblast Growth Medium 2 kit, with 5% CO2. ２ They were cultured at 37°C.

[0046] Western blot cells were seeded in a 6-well plate. Two days after seeding, extracellular secretions were added to a final concentration of 5%, and sEVs and m / LEVs were added in amounts equivalent to 5% of extracellular secretions. The cells were then exposed to 37°C and 5% CO2. ２ The cells were incubated for 8 hours under the following conditions. Torin-2 was added to the cells along with extracellular secretions or EVs to a final concentration of 250 nmol / L, and incubated at 37°C in 5% CO2. ２The cells were incubated under the following conditions for 8 hours. After incubation, the cells were washed with PBS and lysed with a lysis solution containing the complete, Mini protease inhibitor cocktail (#4693124001 Roche) (1% NP-40, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, EDTA 1 mM), and left to stand on ice for 30 minutes. The cell lysate was centrifuged at 20,380 G for 20 minutes at 4°C, and the supernatant was collected. To the recovered supernatant, 25% volume of SDS sample buffer (250 mM Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, 0.02% BromoPhenol Blue) and 5% volume of β-mercaptoethanol were added, mixed, and incubated at 65°C for 30 minutes. This mixture was used as the SDS sample and separated by SDS-PAGE.

[0047] SDS-PAGE gels were prepared using Invitrogen® Bolt® Bis-Tris Plus Gels. The separated gels were transferred to a 0.2 μm PVDF membrane using Invitrogen® Bolt® iBlot3 Westernblot Transfer System. The transferred PVDF membrane was incubated at room temperature for 1 hour using Blocking One (#03953-95 Nacalai Tesque) for blocking. After blocking, the primary antibody was incubated on the proteins on the PVDF membrane. After washing three times for 10 minutes with Tris-buffered saline (T-TBS) containing 0.1% Tween®-20, the secondary antibody was incubated at room temperature for 1 hour. After incubation of the secondary antibody, the solution was washed three times with T-TBS for 10 minutes each. Both the primary and secondary antibodies were diluted with a 20-fold dilution of blocking solution before use.

[0048] The washed PVDF membranes were incubated with either ECL plus westernblotting detection reagent (32132 Pierce) or ECL select westernblotting detection reagent (RPN2232 Cytiva) at room temperature for 5 minutes. The resulting signals were detected using the BioRad ChemiDoc® MP Imaging system.

[0049] ・Use of primary antibody ATP5A ミトコンドリアマーカー (Abacm #ab14848) LC3 (MBL #PM036) p62 (SQSTM1) (MBL #PM045) GAPDH (14C10) (Cell Signaling Technology #2118) ULK-1 ((D8H5) (Cell Signaling Technology #8054T)ホスホULK1(Ser757)(Cell Signaling Technology #6888T) p70 S6キナーゼ(49D7)(Cll #2708T) ホスホp70 S6キナーゼ(Thr389)(108D2)(Cell Signaling Technology #9234T) mTOR (7C10) (Cell Signaling Technology #2983T) TOR Technology #5536T) BiP Antibody (Cell Signaling Technology #3183S) 4E-BP1 (53H11) (Cell Signaling Technology #9644T) ホスホp44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling Technology #4370T) ホスホp38 MAPK (Thr180 / Tyr182) (Cell Signaling Technology #9211S) ホスホMEK1 / 2 (Ser217 / 221) (Cell Signaling Technology #9121S)

[0050] Secondary antibody: anti-USAgi IgG, HRP-linked antibody (Cell Signaling Technology #7074S) Anti-USAgi IgG, HRP-linked antibody (Cell Signaling Technology #7076S)

[0051] Protein assay: Protein quantification was performed using Pierce® BCA Protein Assay Kits (#23225 Thermo Scientific®). Extracellular secretions were quantified using dH ２ Samples were prepared by diluting the solution 5-fold, 10-fold, and 20-fold with O and transferred to a 96-well microplate. Using the BSA standard solution provided with the kit, BSA standard dilution samples were prepared and transferred to a 96-well microplate in the same manner as the samples. BCA Reagent A and BCA Reagent B provided with the kit were mixed to prepare the test solution. Eight times the volume of the test solution of the sample to be measured was added to each well of the 96-well microplate and mixed with a plate shaker for 30 seconds. The plate was covered and incubated at 37°C for 30 minutes. After incubation, the plate was returned to room temperature and the absorbance at 562 nm was measured using a microplate reader. A Tecan INFINITE M PLEX (Tecan) microplate reader was used.

[0052] (Results) SH-SY5Y cells were treated with extracellular secretions, and the results were observed. To more clearly evaluate the effect on activation of the mTOR pathway, Torin2, an inhibitor of mTOR kinase activity, was used as appropriate. Torin2 is known to bind to the ATP binding pocket of the mTOR complex and inhibit ATP from binding to the mTOR kinase, and is known to inhibit both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As shown in Figures 3-5, ES-S showed the function of activating the mTOR pathway for phosphorylation of any of the mTOR pathway molecules, including mTOR, ULK1, S6K1, and 4E-BP1, but no activation of the mTOR pathway was observed with sEVs. As shown in Figure 6 for ERK1 / 2, similarly for the MAPK pathway, ES-S showed activation, while sEVs did not. These results indicate that the supernatant of extracellular secretions acts as an accelerator, activating the mTOR and MAPK pathways, while sEVs act as a brake, suppressing the activated mTOR and MAPK pathways to some extent. Similar experiments conducted with varying concentrations of extracellular secretions, ES-S, and sEVs also suggested the above trend (results not shown).

[0053] Furthermore, when EXO-Prep (Cosmo Bio) was used to separate the EV fraction, it was similarly observed that the supernatant of extracellular secretions activated the mTOR pathway, and EVs suppressed the activated mTOR pathway to some extent (Figure 7). The lanes in Figure 7 are as follows: 1. Untreated control 2. Addition of inhibitor BafA1 only 3. Addition of extracellular secretions 4. Addition of EXO-Prep extract

[0054] (Example 3: Relationship with the Biological Function of Extracellular Secretions) The mTOR pathway and the MAPK pathway are associated with various biological functions. Using SH-SY5Y cells, we investigated how extracellular secretions affect biological functions.

[0055] (Effects on Autophagy) To investigate the effects of extracellular secretions on autophagy, LC3 punctae and p62 were examined. The results are shown in Figure 8. Treatment with extracellular secretions reduced the amount of LC3 punctae in cells and the Western blot band of LC3, and accumulation of p62, an autophagy substrate, was observed. This suggests that extracellular secretions tend to suppress autophagy.

[0056] (Effects on mitochondria) To investigate the effects of extracellular secretions on mitochondria, Mitotracker Red CM-H ２ ROS was used. This reagent fluoresces when oxidized in mitochondria, and therefore serves as an indicator of mitochondrial membrane potential (metabolic activity). The results are shown in Figure 9. Treatment with extracellular secretions enhanced mitochondrial signaling. Extracellular secretions tended to improve mitochondrial activity.

[0057] (Effects on cellular stress) To investigate the effects of extracellular secretions under cellular stress conditions, tunicamycin was used to mimic endoplasmic reticulum stress. Tunicamycin suppresses mTORC1 activity via the PERK pathway by phosphorylation of eIF2α and activation of AMPK. The results are shown in Figure 10. Treatment with extracellular secretions resulted in increased S6K phosphorylation under endoplasmic reticulum stress conditions, suggesting that extracellular secretions have an effect of alleviating endoplasmic reticulum stress.

[0058] (Discussion) The above experiments suggest that the extracellular secretions of Example 1 interfere with the mTOR and MAPK pathways, affecting autophagy, mitochondrial activity, and cellular stress. Furthermore, it was shown that the supernatant of the extracellular secretions activates the mTOR and MAPK pathways, while small extracellular vesicles (sEVs) suppress the activated mTOR and MAPK pathways, thereby regulating their activity and achieving appropriate mTOR and MAPK activation. Due to this appropriate mTOR and MAPK activation, the extracellular secretions of this disclosure are expected to be useful in treating or preventing various diseases. In addition, since the effect was observed in the nerve cell SH-SY5Y, the extracellular secretions of this disclosure may also be used in treating or preventing neurological diseases. In fact, the activation (phosphorylation) of S6K, which was also tested in the above examples, is known to be a cause of diabetes and neurological diseases.

[0059] (Example 4: Reduction of aging by extracellular secretions) As described above, extracellular secretions showed effects on the mTOR pathway and the MAPK pathway, and regulated their biological functions. Based on these molecular behaviors, an effect on aging was expected, so we investigated the effect of extracellular secretions on aging.

[0060] (Establishment of senescent cells) Senescent cells were established by passage 25 times or more in 10φ dishes using two kits of fibroblast growth medium (Fibroblast Growth Medium 2) (Takara Bio) HDF (human dermal fibroblast) cells (purchased from Takara Bio) as fibroblast growth medium. An increase in β-gal activity, a senescence marker, was observed from passage 16 (P16) onwards, and stable high β-gal activity was observed from passage 25 or more.

[0061] The measurements in this embodiment were performed as follows.

[0062] (β-Gal measurement) Cellular senescence was measured using the Cellular Senescence Plate Assay Kit - SPiDER-βGal (Dongrentang). 3.0 × 10⁶ cells were used in a 96-well black plate. ３ Seed cells in 100 μL of water at 37°C and 5% CO2.２ Incubate for 24 hours, then replace with medium containing reagent diluted with D-MEM, and incubate at 37°C and 5% CO2. ２ The cells were incubated for 72 hours. To measure the cell count, 100 μL of Working solution was added to each well. The cells were incubated in an incubator at 37°C and 5% CO2. ２ The cells were incubated for 30 minutes. The supernatant was removed, and 100 μL of dilution buffer was added to each well. Fluorescence was measured using a fluorescence plate reader (Bottom to Top, excitation wavelength: 350 nm, emission wavelength: 461 nm). After counting the cells, the supernatant was aspirated, washed once with 100 μL of 1×PBS, 50 μL of lysis buffer was added, and the cells were incubated at room temperature for 10 minutes. 125 μL of DMSO was added to a tube containing SPiDER-βGal to prepare the SPiDER-βGal DMSO stock solution. The SPiDER-βGal DMSO stock solution was diluted 10-fold with assay buffer to prepare the SPiDER-βGal working solution. 50 μL of the SPiDER-βGal working solution was added to each well, and the cells were incubated at 37°C for 24 hours. 100 μL of stop solution was added to each well. Fluorescence was measured using a fluorescence plate reader (Bottom to Top, excitation wavelength: 535 nm, emission wavelength: 580 nm).

[0063] (Cell proliferation WST-8 assay) Measurement was performed using Cell Counting Kit-8 (Dongrentang). 5.0 × 10⁶ cells were placed in a 96-well plate. ３ Seed cells at a concentration of 100 μL per cell, at 37°C and 5% CO2. ２ Incubate for 24 hours, then replace with medium containing reagent diluted with D-MEM, and incubate at 37°C and 5% CO2. ２ The samples were incubated for 48 hours. After adding WST-8 and incubating for 2 hours, measurements were taken using a plate reader (450 nm).

[0064] (ATP assay) ATP activity was measured using the ATP Assay Kit-Luminescence (Dongrentang). Cells were diluted with D-MEM(-) and placed in a 96-well plate at a rate of 1 × 10⁶ cells. ４Cells were seeded at a rate of 100 μL / cell, washed with D-MEM(-), and then 100 μL of D-MEM(-) was added to each well. 100 μL / well of ATP standard solution was added to each well. Working solution was added to each well. The samples were centrifuged in a plate shaker at approximately 400 rpm for 2 minutes. The plates were placed in a plate reader and incubated at 25°C for 10 minutes. Relative luminescence (RLU) was measured, and the ATP concentration in the sample was calculated using a calibration curve.

[0065] (NAD / NADH assay, doubling time measurement) The NAD / NADH ratio was measured using the NAD / NADH Assay Kit-WST (Tongrentang). 3.0 × 10⁻¹⁰ ５ Cells were seeded in each 10Φ petri dish. The culture medium was changed with either unadded (control) or reagent-added medium, and the cells were incubated. The cells were harvested, the number of cells was counted, and the cells were centrifuged at 1000 rpm for 3 minutes. After removing the supernatant, the cells were suspended in 1 mL of PBS, transferred to a 1.5 mL tube, and centrifuged at 300 g for 5 minutes. 500 mL of PBS was added, pipetted, 300 μL of NAD / NADH extraction buffer was added, and the cells were lysed by pipetting, then centrifuged at 12,000 g for 5 minutes. 250 μL of the supernatant was transferred to an MWCO 10K filtration tube and centrifuged at 12,000 g for 10 minutes. 80 μL of the filtrate was transferred to two 1.5 mL tubes, each used as a sample for measuring total NAD+ / NADH and NADH levels, respectively. Samples for NADH level measurement were incubated at 60°C for 60 minutes, while samples for NAD+ / NADH level measurement were treated with ice. After incubation, the samples were allowed to return to room temperature. 100 μL of NAD / NADH control buffer was added to each tube. Standard solutions and samples were added to 96-well clear plates at 50 μL / well. 50 μL of working solution was added, and the plates were incubated at 37°C for 60 minutes. Absorbance was then measured at 450 nm using a plate reader. The cell doubling time under each culture condition was calculated based on the number of cells before and after incubation.

[0066] (DNA Damage Measurement) DNA damage was measured using the DNA Damage Detection Kit - γH2AX (Red) (Dongrentang). Cells were placed in an 8-well chamber slide at a density of 1.0 × 10⁶. ４ Seed at a cell / well density at 37°C and 5% CO2. ２ Incubate for 24 hours, then replace with medium containing reagent diluted with D-MEM, and incubate at 37°C and 5% CO2. ２ The cells were incubated for 48 hours. The supernatant was removed, washed twice with PBS, and 100 μL of 250 mM HEPES (pH 7.4) solution containing 4% PFA and 0.1% Triton® X-100 was added. The cells were allowed to stand at room temperature for 5 minutes. The cells were washed twice with PBS, and 100 μL of PBS solution containing 1% Triton® X-100 was added. The cells were allowed to stand at room temperature for 20 minutes. The cells were washed twice with PBS, and 100 μL of blocking solution was added. The cells were allowed to stand at room temperature for 20 minutes. The cells were washed twice with PBS, and 100 μL of anti-γH2AX antibody staining solution was added. The cells were incubated for 1 hour. The cells were washed twice with PBS, and 100 μL of secondary antibody staining solution was added. The cells were incubated for 1 hour, washed twice with PBS, and observed under a fluorescence microscope.

[0067] (Experiment and Results) As described above, the cells established showed an increase in β-gal activity, a senescence marker, from passage 16 (P16) onwards (Figure 11). Therefore, cells from passages 1 to 10 (P1 to 10) were designated as young cells, and cells from passages 25 to 40 (P25 to 40) were designated as senescent cells and used in subsequent experiments.

[0068] To demonstrate the effect of the extracellular secretions of Example 1, OC-01, a human adipose-derived stem cell culture supernatant (hereinafter also referred to as the "comparative product"), was obtained as a control. When small-sized extracellular vesicles (sEVs) were extracted from the same volume of extracellular secretions of Example 1 and the comparative product, as shown in Example 1, it was found that the extracellular secretions of Example 1 contained more sEVs than the comparative product (Figure 12). When comparing all EVs, the extracellular secretions of Example 1 also contained more EVs than the comparative product. The total EVs of the comparative product were approximately 5.11 x 10⁶. ９The density was approximately 135.6 ± 3.0 nm, with a mode of approximately 98.9 ± 3.3 nm and an average of approximately 135.6 ± 3.0 nm.

[0069] The effects of the extracellular secretions from Example 1 and the comparative product on young and aged cells were tested. In each test, the comparative product was added at a concentration of 5% of the culture medium, and the extracellular secretions (ES) from Example 1 were added at a concentration of 5% or 10% of the culture medium.

[0070] Figures 13-17 show the effects of the extracellular secretion (ES) of Example 1 on β-gal activity, cell proliferation, doubling time, and mitochondrial activity in senescent cells. The extracellular secretion (ES) of Example 1 was able to reduce the elevated β-gal activity in senescent cells in a dose-dependent manner, whereas no such effect was observed with the comparative product (Figure 13). When cell proliferation was measured by WST-8 activity, both the extracellular secretion (ES) of Example 1 and the comparative product were observed to activate cell proliferation, but the effect of the extracellular secretion of Example 1 was more potent (Figure 14). A similar trend was observed in doubling time, with the extracellular secretion (ES) of Example 1 activating senescent cells and shortening the doubling time more potently than 1 mM NMN (nicotinamide mononucleotide) (Oriental Yeast Co., Ltd.) (Figure 15). Mitochondrial activity was evaluated based on ATP levels (Figure 16) and NAD+ levels (Figure 17). The extracellular secretion (ES) of Example 1 enhanced mitochondrial activity more potently than the comparative product, was more potent than 1 mM NMN, and was equivalent to 1 mM ALA (5-aminolevulinic acid; FUJIFILM (CAS RN: 5451-09-2)). ALA is a drug known to be taken up by mitochondria and biosynthesized as protoporphyrin IV, thereby increasing mitochondrial activity.

[0071] γH2AX is a marker of DNA damage, and it was increased in aged cells compared to young cells (Figure 18). Doxorubicin (Dox) (Funakoshi; CAS: 25316-40-9), which is known to artificially act on the G2 / M phase of the cell cycle and induce cellular senescence, increased γH2AX staining spots in young cells, but did not have a significant effect on aged cells, indicating that aged cells already have reduced cell proliferation capacity (Figures 19, 20). When the effect of the extracellular secretion from Example 1 (added to the culture medium at a 10% concentration) on DNA damage was evaluated using γH2AX, it was observed that the extracellular secretion from Example 1 significantly reduced the increased γH2AX staining spots in aged cells, indicating that it can reduce DNA damage (Figures 19, 20).

[0072] (Discussion) From the above results, it is considered that the extracellular secretion of Example 1 contains many sEVs and can activate senescent cells and restore them from the senescent state, while reducing DNA damage, thus reducing the risk of causing cellular abnormalities such as cancer associated with cell activation. In particular, it is remarkable that the extracellular secretion of Example 1 showed superior anti-aging effects compared to NMN, which is known to have anti-aging effects. Based on the observed anti-aging effects, the extracellular secretion of this disclosure may be applied to the following diseases.

[0073] Senescent cells are known to accumulate in the brain, promote neuroinflammation, and cause Alzheimer's disease and Parkinson's disease, and activation of mTOR has been reported to contribute to improving the pathology of Alzheimer's disease model mice (Baker, D. J., et al. (2011). "Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.", Nature; Ma, T., et al. (2010). "Dysregulation of the mTOR pathway mediates impairment of synaptic plasticity in a mouse model of Alzheimer's disease.", PLoS One). Therefore, based on the effect on the nerve cell SH-SY5Y shown in the above examples, the extracellular secretions of this disclosure may be used in the prevention or treatment of Alzheimer's disease and Parkinson's disease.

[0074] It has been reported that prolonged hyperglycemia leads to the accumulation of senescent cells and worsens insulin resistance (Tchkonia, T., et al. (2013). "Cellular senescence and the senescent secretory phenotype: therapeutic opportunities." The Journal of Clinical Investigation). Therefore, the extracellular secretions of this disclosure may be used to prevent or treat diabetes (particularly type 2 diabetes).

[0075] Senescent cells have been reported to promote vascular hardening and inflammation (Childs, B. G., et al. (2016). "Senescent intimal foam cells are deleterious at all stages of atherosclerosis." Science). Therefore, the extracellular secretions of this disclosure may be used to prevent or treat cardiovascular diseases (atherosclerosis, hypertension, myocardial infarction, and stroke) by reducing cellular senescence.

[0076] It has been reported that senescent cells accumulate in the lungs due to smoking or air pollution, contributing to the progression of chronic obstructive pulmonary disease (COPD) (Barnes, P. J., et al. (2009). "Cellular senescence as a mechanism and target in chronic lung diseases." Nature Reviews Drug Discovery). Therefore, the extracellular secretions of this disclosure may also be used to prevent or treat COPD.

[0077] Senescent cells present in bone or joints have been reported to secrete inflammatory cytokines that promote inflammation. Therefore, the extracellular secretions described herein may also be used to prevent or treat arthritis and bone diseases (Jeon, O. H. et al. (2017). “Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment.” Nature medicine, 23(6), 775-781.).

[0078] This disclosure provides extracellular secretions useful for the treatment of various diseases.

Claims

1. Approximately 6×10 １０ Extracellular secretions containing small extracellular vesicles (sEVs) of a concentration of 1 or more cells / mL.

2. Extracellular secretions containing extracellular vesicles (EVs) with a particle size distribution having a mode of approximately 100 to 120 nm.

3. The extracellular secretion according to claim 1 or 2, wherein the proportion of small-sized extracellular vesicles (sEVs) among all extracellular vesicles is approximately 90% or more.

4. The extracellular secretion according to claim 1 or 2, which is obtained from the culture supernatant of mesenchymal stem cells.

5. The extracellular secretion according to claim 4, obtained from the culture supernatant of mesenchymal stem cells cultured in a medium containing sodium bicarbonate and having a pH of about 8.0 to about 8.

5.

6. Compositions comprising extracellular secretions for treating or preventing aging or age-related diseases.

7. The composition according to claim 6, wherein the aging-related disease includes Alzheimer's disease or Parkinson's disease.

8. The composition according to claim 6, wherein the aging-related disease includes diabetes.

9. The composition according to claim 6, wherein the aging-related disease includes cardiovascular disease.

10. The composition according to claim 6, wherein the aging-related disease includes chronic obstructive pulmonary disease (COPD).

11. The composition according to claim 6, wherein the aging-related disease includes arthritis and bone disease.

12. A composition containing extracellular secretions for the regulation of mTOR activity or MAPK activity.

13. The composition according to any one of claims 6 to 12, wherein the extracellular secretion is obtained from the culture supernatant of mesenchymal stem cells.

14. The composition according to any one of claims 6 to 12, wherein the extracellular secretion is the extracellular secretion according to any one of claims 1 to 5.

15. An extracellular secretion according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 14, for injection.

16. A method for preparing a supernatant fraction or extracellular vesicle fraction of an extracellular secretion, comprising the step of separating the extracellular secretion described in any one of claims 1 to 5 into a supernatant fraction and an extracellular vesicle fraction.

17. A supernatant fraction, an extracellular vesicle fraction, or a combination comprising the supernatant fraction and the extracellular vesicle fraction obtained by the method described in claim 16.

18. A method for examining extracellular secretions, comprising the steps of: incubating human cultured cells in a culture medium containing a predetermined amount of the extracellular secretions; and measuring the phosphorylation of one or more of mTOR, S6K1, ULK1, 4E-BP1, and ERK1 / 2 in cells incubated with the extracellular secretions, wherein the extracellular secretions are deemed acceptable if the phosphorylation is elevated compared to a control condition without extracellular secretions and is lower compared to the extracellular secretions from which EVs have been removed.

19. A method for testing extracellular secretions, comprising the steps of: incubating human cultured senescent cells in a culture medium containing a predetermined amount of the extracellular secretions; and measuring one or more of the following items in the cells incubated with the extracellular secretions: β-Gal activity, cell proliferation activity, cell doubling time, mitochondrial activity, and DNA damage, wherein the extracellular secretions are deemed acceptable if the measured values ​​for the items show improvement in indicators of senescent cells compared to a control condition without extracellular secretions.

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