Brms and use thereof

Abstract

The present disclosure provides BRMs useful for the treatment of various diseases. In one embodiment, the present disclosure provides BRMs comprising small extracellular vesicles (sEVs) at a concentration of approximately 6×1010 vesicles / mL or more. In one embodiment, the present disclosure provides BRMs comprising small extracellular vesicles (sEVs) having a particle size distribution in which a mode particle size is approximately 100 nm to approximately 120 nm. In one embodiment, the BRMs are obtained from a culture supernatant of mesenchymal stem cells. In one embodiment, aging is treated with BRMs.

Description

BRMs and their use

[0001] This disclosure relates to BRMs (Biological Response Modifiers) and their use.

[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 BRMs useful for treating diseases and have completed the present invention.

[0004] This disclosure provides, for example, the following items: (Item 1) (a) Approximately 6 x 10 １０ (b) BRMs comprising small extracellular vesicles (sEVs) of 1 / mL or more, or (b) prepared by removing extracellular vesicles (EVs) from the culture supernatant of stem cells. (Item 2) A composition comprising any of the above BRMs for treating or preventing aging or age-related diseases. (Item 3) A composition comprising any of the above BRMs for increasing the proliferative capacity of cells. (Item 4) A composition comprising any of the above BRMs for treating or preventing aging. (Item 5) A composition comprising any of the above BRMs for enabling the proliferation of aged cells. (Item 6) A composition comprising any of the above, wherein the age-related disease comprises Alzheimer's disease or Parkinson's disease. (Item 7) A composition comprising any of the above, wherein the age-related disease comprises diabetes mellitus. (Item 8) A composition comprising any of the above, wherein the age-related disease comprises cardiovascular disease. (Item 9) The aging-related disease is a composition of any of the above items, including chronic obstructive pulmonary disease (COPD). (Item 10) The aging-related disease is a composition of any of the above items, including arthritis and bone disease. (Item 11) A composition for modulating mTOR activity or MAPK activity, comprising any of the above items' BRMs. (Item 12) A composition for use in cell culture, comprising any of the above items' BRMs. (Item 13) A composition of any of the above items for injection. (Item 14) The BRMs are approximately 6 × 10 １０A BRM containing small extracellular vesicles (sEVs) of 10 or more cells / mL, which is any of the compositions listed above. (Item 15) Approximately 6 × 10 １０BRMs according to any of the above items, containing small extracellular vesicles (sEVs) of 100 pcs / mL or more. (Item 16) BRMs according to any of the above items, containing extracellular vesicles (EVs) having a particle size distribution with a mode particle size of approximately 100 to approximately 120 nm. (Item 17) BRMs according to any of the above items, in which the proportion of small extracellular vesicles (sEVs) among all extracellular vesicles is approximately 90% or more. (Item 18) BRMs according to any of the above items, prepared by removing extracellular vesicles (EVs) from the culture supernatant of stem cells. (Item 19) BRMs according to any of the above items, obtained from the culture supernatant of mesenchymal stem cells. (Item 20) BRMs according to any of the above items, 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 21) A method for preparing a supernatant fraction or extracellular vesicle fraction of BRMs, comprising the step of separating any of the above BRMs into a supernatant fraction and an extracellular vesicle fraction. (Item 22) A supernatant fraction, an extracellular vesicle fraction, or a combination comprising a supernatant fraction and an extracellular vesicle fraction obtained by the method of Item 21. (Item 23) A method for testing BRMs, comprising the steps of incubating human cultured cells in a medium containing a predetermined amount of the BRMs, and measuring the phosphorylation of one or more of mTOR, S6K1, ULK1, 4E-BP1, and ERK1 / 2 in cells incubated with the BRMs, wherein the BRMs are deemed acceptable if the phosphorylation is elevated compared to a control condition without BRMs and is decreased compared to the BRMs from which EVs have been removed. (Item 24) A method for testing BRMs, comprising the steps of: incubating human cultured senescent cells in a culture medium containing a predetermined amount of the BRMs; and measuring one or more of the following items in the cells incubated with the BRMs: β-Gal activity, cell proliferation activity, cell doubling time, mitochondrial activity, and DNA damage, wherein the BRMs are judged to be acceptable if the measured values ​​for the items are improved in terms of indicators of senescent cells compared to a control condition without BRMs.

[0005] The BRMs disclosed herein are useful for treating various diseases.

[0006] This section outlines the procedure for isolating small-sized extracellular viable (EV) fractions (sEVs) from BRMs. The particle size distribution of all EVs contained in the BRMs is shown with error bars. The left side shows the Western blot results for phosphorylated mTOR and GAPDH under various sample conditions under Torin treatment (Torin-2: 250 nM for 8 hours). The right side shows the quantitative results for phosphorylated mTOR under various sample conditions (shown as relative intensity to GAPDH). The numbers indicate the following sample conditions: 1: Untreated (Control), 2: BRMs, 3: Small-sized EV fraction (sEVs), 4: BRMs-S (centrifugation supernatant). p70 This section shows the Western blot results for phosphorylation of S6 kinase (S6K). The left side shows the Western blot results for S6K protein expression and phosphorylation under various sample conditions under Torin treatment (Torin-2: 250 nM for 8 hours). The right side shows the quantitative results of phosphorylated S6K under each sample condition (shown as relative intensity to GAPDH). The numbers indicate the following sample conditions: 1: Untreated (Control), 2: BRMs, 3: Small-size EV fraction (sEVs), 4: BRMs-S (centrifugation supernatant). The Western blot results for phosphorylation of ULK1 (left) and 4E-BP1 (right) under each sample condition under Torin treatment (Torin-2: 250 nM 8 hours) are shown. The numbers indicate the following sample conditions: 1: Untreated (Control), 2: BRMs, 3: Small-size EV fraction (sEVs), 4: BRMs-S (centrifugation supernatant). In 4E-BP1, the band appears higher in the upper part as phosphorylation increases. The Western blot results for phosphorylation of ERK1 / 2 are shown. The numbers indicate the following sample conditions: 1: Untreated (Control), 2: BRMs, 3: Small-size EV fraction (sEVs), 4: BRMs-S (centrifugation 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 + BRMs, 4: EXO-Prep extracted EVs. A) Microscopic images of the LC3 staining trend of cells cultured with or without BRMs added along with BafA1 are shown. Puncta formation is observed under the condition with BRMs added. B) The number of cells and total number of cells showing puncta formation are shown under the culture conditions with or without BRMs added. C) Western blot results showing p62 and LC3 expression under the culture conditions with or without BafA1 and BRMs added are shown. Mitotracker Red CM-H of cells cultured under each culture condition. ２ The staining results using ROS are shown. When BRMs 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 BRMs 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 BRMs of 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 BRMs of Example 1 contain more particles. The effect of BRMs 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, BRMs 5%, BRMs 10%. * indicates a statistically significant difference of p < 0.05. This shows the effect of BRMs 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, BRMs 5%, BRMs 10%. * indicates a statistically significant difference of p < 0.05. This shows the effect of BRMs 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, BRMs 5%, BRMs 10%. This shows the effect of BRMs 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, BRMs 5%, BRMs 10%. * indicates a statistically significant difference of p < 0.05. This shows the effect of BRMs 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, BRMs 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 BRMs or Dox (doxorubicin). This shows the number of γH2AX staining spots when young and aged cells were treated with BRMs or Dox (doxorubicin).This shows a comparison of cell counts when P42 senescent cells were cultured for 7 days under conditions without additives (Control), BRMs, or BRMs-S. It also shows the WST-8 assay results when P35 senescent cells were cultured for 2 days under conditions without additives (Control), BRMs, BRMs-S, or NMN. The vertical axis shows the measured absorbance (450 nm) for each condition, excluding the Blank value.

[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, “BRMs (Biological Response Modifiers)” means substances produced by cells that have some biological function, or compositions containing such substances. BRMs may be compositions containing substances produced by cells and released extracellularly during cell culture (sometimes abbreviated as “extracellular secretions” or “ES” in this specification). Typically, BRMs are preparations of the supernatant or components (extracellular vesicles, proteins, etc.) of a cell culture that does not contain cells themselves. BRMs are also obtained by removing specific 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] (BRMs) In one aspect, the present disclosure provides BRMs. In one embodiment, the BRMs comprise the culture supernatant of mesenchymal stem cells or components thereof. Therefore, the BRMs may contain medium components used for cell culture (e.g., salts, buffers, growth factors, vitamins, minerals, cytokines, etc.). In one embodiment, the BRMs do not contain cells. Cells can be easily removed from the culture supernatant using a filter or the like.

[0014] Typically, the BRMs of the present disclosure are those with adjusted cell culture supernatant or its concentration of extracellular vesicle components. As shown in the examples, in the culture supernatant of mesenchymal stem cells, extracellular vesicles and other components were found to have different functions in mTOR activity and MAPK activity. Therefore, by adjusting the concentration of extracellular vesicle components in the culture supernatant, the mTOR activity and MAPK activity of BRMs 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 easily concentrate or reduce extracellular vesicles with minimal impact on biological materials such as proteins contained in BRMs.

[0015] In one embodiment, the BRMs are characterized by small-sized extracellular vesicles (sEVs). Excessive mTOR activation can be inhibited by small-sized extracellular vesicles (sEVs). In one embodiment, the BRMs contain １０ at least about 1×10 １０ per mL or more, at least about 2×10 １０ per mL or more, at least about 5×10 １０ per mL or more, at least about 5×10 １１ per mL or more, at least about 1×10 １１ per mL or more, and / or at least about 5×10 １０ per mL or less, at least about 1×10 １１ per mL or less, at least about 2×10 １１ per mL or less, at least about 5×10 １１ per mL or less, or at least about 1×10 １２ small-sized extracellular vesicles (sEVs) per mL or less.

[0016] In one embodiment, the small-sized extracellular vesicles (sEVs) in BRMs have a particle size most frequent 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-sized extracellular vesicles (sEVs) in BRMs have a particle size distribution with a full width at half maximum of about 40 to about 90 nm, about 50 to about 80 nm, about 55 to about 75 nm, or about 60 to about 70 nm.

[0018] In one embodiment, the proportion of small-sized extracellular vesicles (sEVs) among all extracellular vesicles in the BRMs of the present invention can be 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.

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

[0020] (Preparation of BRMs) In one embodiment, BRMs can 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, neurons, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, osteocytes, chondrocytes, germ line cells or iPS cells, and preferably are derived from adipocytes.

[0021] Cell culture conditions can affect the components of BRMs (such as extracellular vesicles). In one embodiment, the BRMs of this disclosure are prepared from the culture supernatant of mesenchymal stem cells cultured at pH levels of approximately 7.0 to 10.0, 7.0 to 9.5, 7.0 to 9.0, 7.0 to 8.5, 7.5 to 10.0, 7.5 to 9.5, 7.5 to 9.0, 7.5 to 8.5, 8.0 to 10.0, 8.0 to 9.5, 8.0 to 9.0, 8.0 to 8.5, 8.5 to 10.0, or 8.5 to 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 BRMs 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 disclosure provides a method for separating cell culture supernatant into an extracellular vesicle fraction and a supernatant fraction. The fraction containing a higher concentration of extracellular vesicles compared to the original cell culture supernatant is called the extracellular vesicle fraction, and the fraction containing a lower concentration of extracellular vesicles is called the supernatant fraction. Methods such as centrifugation, sucrose density gradient ultracentrifugation, tangential flow filtration, and size exclusion chromatography can be used for separation. The disclosure also provides the extracellular vesicle fraction and the supernatant fraction. In one embodiment, the extracellular vesicle fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations 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 2000% of the cell culture supernatant. In one embodiment, the supernatant fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations of up to approximately 90%, 80%, 50%, 20%, 15%, 10%, 7%, 5%, 2%, 1.5%, or 1% of the cell culture supernatant. The disclosure also provides combinations of the extracellular vesicle fraction and the supernatant fraction. While BRMs not separated into the extracellular vesicle fraction and the supernatant fraction may be beneficial by resulting in moderate activation of the mTOR pathway, it may also be beneficial to use the extracellular vesicle fraction and the supernatant fraction separately in regulating the activation of the mTOR or MAPK pathway.

[0024] In a typical embodiment, the BRMs of this disclosure are not exosome concentrates such as purified exosome preparations, but rather liquids from which cells, extracellular matrix, and their fragments have been removed from a cell culture, while retaining most of the other components of the cell culture in amounts equivalent to those of the original cell culture. In one embodiment, the BRMs of this disclosure are prepared without a step of concentrating extracellular vesicles after removing cells from the culture (e.g., by centrifugation). In certain embodiments, a supernatant fraction prepared by removing at least some extracellular vesicles from a cell culture supernatant (e.g., prepared after removing cells from a cell culture) may be suitably used. In one embodiment, the extracellular vesicles contained in the BRMs are approximately 1 × 10⁶ ６ pcs / mL or less, approximately 1×10 ５ pcs / mL or less, approximately 1×10 ４ pcs / mL or less, approximately 1×10 ３ Less than or equal to 1 x 10 cells / mL ２ The number of particles per mL is less than or equal to 1 / mL.

[0025] In one embodiment, the disclosure provides a method for preparing a composition comprising: culturing mesenchymal stem cells to obtain a mesenchymal stem cell culture; removing cells from the mesenchymal stem cell culture to obtain BRMs; and preparing the BRMs into a composition. The step of preparing the BRMs into a composition does not require altering the components of the BRMs and may include, for example, dispensing the BRMs into containers. In one embodiment, the method for preparing a composition comprising BRMs does not include a step of concentrating extracellular vesicles after removing cells from the culture (e.g., centrifugation).

[0026] In one embodiment, the present disclosure provides a method for evaluating the mTOR pathway and / or MAPK pathway activation ability of BRMs (e.g., cell culture supernatant). Since the performance of BRMs can vary due to various factors in culture, a method for evaluating the performance of BRMs may be useful in order to ensure the usefulness of BRMs. In one embodiment, the BRMs of the present disclosure may have a higher mTOR pathway and / or MAPK pathway activation ability when sEVs are removed by centrifugation or other means compared to when this removal operation is not performed.

[0027] (Composition) In one embodiment, the Disclosure provides a composition comprising the BRMs 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.

[0028] 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.

[0029] The amount of the composition disclosed herein may vary depending on the nature of the disorder or condition being treated or prevented, but can be determined by standard clinical techniques based on the description herein for those skilled in the art. In some cases, in vitro assays may be used to assist in identifying the optimal dosage range. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the attending physician and the circumstances of each patient. The dosing interval is not particularly limited, but may be once or twice every 1, 7, 14, 21, or 28 days, or once or twice every two of these intervals. The dosage, dosing interval, and method of administration may be appropriately selected depending on the patient's age, weight, symptoms, etc.

[0030] The administration routes of the compositions described herein may include, for example, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intraparenchymal, and epidural administration. Local administration is also permitted, but even local diseases can be treated by intravenous administration.

[0031] The compositions of the present disclosure may be provided as kits. In one embodiment, the present disclosure provides a drug pack or kit comprising one or more containers filled with one or more components that may be added to the compositions of the present disclosure. Optionally, such containers may also include information indicating authorization by a government agency for manufacture, use or sale for human administration, in a form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceutical or biological products.

[0032] (Applicable Diseases) In one embodiment, the BRMs or compositions of the Disclosure are used to treat or prevent aging, or to modulate mTOR (e.g., mTORC1) and / or MAPK. The Disclosure also provides methods for using the BRMs or compositions of the Disclosure in this manner. From the data shown in the Examples, the BRMs of the Disclosure are found to be particularly useful in treating or preventing aging or age-related diseases.

[0033] Aging can refer to both cellular aging and biological aging. Cellular aging is typically a state in which cells maintain viability and metabolic activity, but cell division has stopped and proliferative capacity has been lost. 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 BRMs 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. In this specification, senescent cells include senescent cells (which may also be referred to as low-senescent cells) and high-senescent cells.

[0034] Severely aged cells refer to cells that cannot proliferate under normal culture conditions for their cell type (they do not increase in number even when cultured). Severely aged cells may have β-galactosidase activity that is approximately 5 times, 7 times, or 10 times higher than the average β-galactosidase activity of the same cell type obtained from young subjects (e.g., teenagers or people in their 20s in humans) (e.g., β-galactosidase activity measured under the same conditions as the measurement conditions in the examples). Aged cells refer to senescent cells that are not severely aged cells, and may have β-galactosidase activity that is approximately 2 times or higher than the average β-galactosidase activity of the same cell type obtained from young subjects.

[0035] While the normal culture conditions for each cell type can be appropriately determined by those skilled in the art, the following are standard examples of conditions for obtaining and culturing primary cultured cells (normal cells). • (Preparation of reagents and equipment) Select a basal medium suitable for the target cell type (e.g., DMEM, RPMI-1640, Neurobasal, etc.), add L-glutamine (e.g., 2-4 mM) and / or essential amino acids or vitamins, add 5-20% sterile, heat-inactivated FBS depending on the cell type, and add antibiotics such as penicillin, streptomycin, or anti-micerium preparations (additives to prevent or detect contamination by fungi (especially molds and yeasts)) as needed. Filter the medium through a 0.22 μm filter and store at 37°C / 5% CO2. ２ Keep warm. Autoclave culture flasks, dishes, tubes, and pipette tips, or use pre-made sterile items. Wipe instruments used in the clean bench with 70% ethanol and briefly irradiate with UV light. Sterilize enzyme solutions and PBS. If necessary, sterilize the culture surface with poly-L-lysine (e.g., 0.01–0.1 mg / mL) and collagen (e.g., 1–50 μg / cm³). ２), it may be coated with laminin or the like. ・(Obtaining cells from tissue) Place the aseptically excised tissue in chilled sterile PBS or cold medium, wash several times with PBS at room temperature or 4°C to remove contamination such as blood and thrombi, remove excess connective tissue, fat, and capsule with sterile scissors / scalpel, and uniformly cut the target area into 1-2 mm square sections. Then, dissolve an enzyme suitable for the tissue (for example, 0.05-0.25% trypsin-EDTA or 20-30 U / mL Papain for nerves, or 0.1-1 mg / mL collagenase for connective tissue) in sterile buffer, add DNase I (10-100 μg / mL) as needed, warm at 37°C, transfer the tissue sections to sterile tubes, add the enzyme solution by 5-10 times the volume, and incubate in a 37°C water bath or under gentle shaking conditions of 50-100 rpm. Digestion time ranges from 10 minutes to several hours depending on the tissue. Gently pipette every 10 minutes to check the degree of dissociation, avoiding excessive shearing. Once sufficiently suspended, collect the digested fluid in FBS-containing medium to neutralize the enzyme (neutralize trypsin with complete medium in a 1:1 to 1:5 ratio, stop Papain with a dedicated inhibitor, etc.). Further dissociate any remaining clumps by gently pipetting or passing through an 18-21G syringe. Filter the resulting suspension through a sterile cell strainer (70 μm or 40 μm) to remove undigested fragments. After filtration, gently wash with cold medium to remove large debris. Transfer the filtered suspension to a suitable container and centrifuge at 300-500 × g for 5-10 minutes at 4°C or room temperature. Carefully remove the supernatant and repeat resuspension and washing with PBS or medium 1-2 times to remove enzymes and debris. Finally, suspend the remaining cells in a suitable medium and use for cell counting. The viability can be evaluated using trypan blue, etc., and the cell count can be measured using a smear / automatic counter or hemocyte meter. (Culture) Dilute the obtained cells to the desired seeding density, gently seed them in a sterile dish or flask, and let them stand at room temperature for several tens of minutes to promote adhesion. Then, while avoiding damage, gently add the culture medium and culture at 37°C and 5% CO2. ２Start culturing in an incubator, and for the first 24 hours, avoid stimuli such as strong agitation, frequent medium changes, and drug additions to allow the cells to recover and adhere. After 24 to 48 hours, check the morphology and adhesion rate with a phase-contrast microscope and perform the first medium change. Thereafter, depending on the cell proliferation status, gently detach the cells with trypsin EDTA or similar at the appropriate timing and subculture. Once the cells reach confluence (80-90%), subculture at 37°C and 5% CO2. ２ The delamination procedure is performed in an incubator. For example, the delamination procedure involves first warming an enzyme (e.g., trypsin EDTA 0.05-0.25%) in a 37°C incubator for several minutes, then using a cell scraper or pipette, gently introducing the enzyme into a portion of the cultured cell layer while carefully delaminating the cell cap and thin layer. While observing the condition under a microscope, the cells are loosened several times by lightly pipetting as needed. Once proper delamination is complete, the enzyme is neutralized by adding culture medium and transferred to another sterile centrifuge tube with complete medium. Then, the cells are centrifuged at 300-500 x g for 5-10 minutes to precipitate. The supernatant is removed, the remaining cells are washed with PBS or culture medium, and centrifuged again to remove excess enzyme and residue. Next, the cells are stained with trypan blue to check viability and concentration, then diluted and gently seeded in a new dish or flask. After seeding, the cells are allowed to stand to promote adhesion and diffusion, and stored at 37°C and 5% CO2. ２ The cells are cultured under these conditions. After 24 to 48 hours, the adhesion and morphology are checked. If good, the culture medium is changed. Then, when the cells approach confluence, enzyme detachment is performed again, and the cells are seeded at a subculturing ratio of 1:3 to 1:6 for long-term continuous culture.

[0036] The culture of SH-SY5Y (derived from human neuroblastoma) and HDF (human dermal fibroblasts) shown in the examples also follows the above-mentioned standard culture conditions. Furthermore, a specific example of the culture of HHSteC (primary cultured human liver astrocytocytes) is presented below. Culture is carried out at 37°C and 5% CO2. ２ The procedure is carried out under these conditions; for static culture, use a medium containing the supplement, and for activation, use a medium without the supplement. * １See ScienCell Research Laboratories' Catalog No. 5301, etc. * ２ Subculturing should be performed using the following procedure, as described in Catalog No. 5352 of ScienCell Research Laboratories. Subculturing should be performed when the culture reaches 90-95% confluence. Poly-L-lysine (2 μg / cm³) should be added the day before the procedure. ２ Prepare a culture vessel coated with ), and allow the culture medium, trypsin, and DPBS to return to room temperature. Next, gently wash the dish with 2 mL of DPBS, then remove the DPBS, add 1 mL of DPBS and 0.05% trypsin EDTA solution, mix well, and detach the cells in a 37°C incubator for about 3 minutes. Once the cells have rounded up and begun to detach, transfer the solution from the dish to a 15 mL centrifuge tube, and gently tap the edge of the dish to detach the cells (confirm under a microscope). Add 1 mL of culture medium and transfer the detached cells to the centrifuge tube, repeating the same procedure until all cells are collected in the centrifuge tube. Centrifuge at 3000 rpm for 3 minutes, remove the supernatant, add another 1 mL of culture medium, and count the number of cells. Next, suspend the cells in 11 mL of culture medium, dispense 4 mL each into three 6 cm dishes, and incubate at 37°C with 5% CO2. ２ Incubate the cells for 2-4 days under these conditions. Avoid excessive damage to the cells during trypsin treatment or pipetting.

[0037] In one embodiment, the BRMs or compositions of this disclosure exhibit: • Higher senescence-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.

[0038] In one embodiment, specific age-related diseases to be treated or prevented include Alzheimer's disease, Parkinson's disease, diabetes (particularly 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 may be treated or prevented by modulation (activation) of the mTOR pathway and / or the MAPK pathway. Furthermore, the BRMs or compositions of this disclosure are expected to treat or prevent aging of the organism (e.g., the human body) composed of cells by treating or preventing cellular aging. In one embodiment, the BRMs or compositions of this disclosure may be used to increase the proliferative capacity of cells (e.g., cells with reduced proliferative capacity). In one embodiment, the treatment or prevention of aging does not include the treatment or prevention of age-related diseases (e.g., any of the examples of age-related diseases described in the specification). In a preferred embodiment, the BRMs or compositions of the present invention may be used to restore proliferative capacity in cells that have lost their ability to proliferate. In one embodiment, the BRMs or compositions of the present invention may be used to enable the proliferation of senescent cells. Progeria and other diseases are thought to involve senescent cells.

[0039] In one embodiment, the BRMs or compositions of the Disclosure are used in cell culture. Since it has been confirmed that the addition of the BRMs of the Disclosure reduces DNA damage and allows even senescent cells to continue proliferating, the BRMs or compositions of the Disclosure can be suitably used in cell culture. For example, the BRMs or compositions of the Disclosure may be added to a culture medium. The cells to be cultured may be any cells, but may be therapeutic cells such as CAR-T cells. The BRMs of the Disclosure can reduce the risk of cancer in cultured cells. Types of cells suitably cultured with the BRMs or compositions of the Disclosure may include stem cells, iPSCs, immortalized cells, and the like.

[0040] (Testing of BRMs) In one embodiment, the disclosure provides a method for testing BRMs and a method for selecting BRMs based on the results. The testing 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.

[0041] In one embodiment, the test may include determining the EV composition contained in the BRMs. 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 BRMs that satisfy one or more of the following conditions can be judged as acceptable: sEVs of 95-125 nm or 100-120 nm (mode), and full width at half maximum (FMAX) of 55-75 nm or 60-70 nm.

[0042] In one embodiment, the test may include determining the effect of BRMs 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 BRMs (and optionally 250 nmol / L of Torin-2) and determining that the phosphorylation of one or more of mTOR, S6K1, ULK1, and 4E-BP1 is increased compared to a control condition without BRMs (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 decreased compared to test BRMs 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 in the amount of phosphorylated protein quantified). BRMs can be judged as acceptable if they satisfy one or more of the following conditions: 1) a decrease of 5 times or less, and 2) 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 test BRMs, the phosphorylation of ERK1 / 2 is increased compared to a control condition without BRMs (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 BRMs 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 protein can be performed by measuring luminescence intensity by immunostaining, ELISA, etc. Control conditions (conditions without BRMs, 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.).

[0043] In one embodiment, the test may include determining the effect on cellular senescence. The test may include: • Incubating human cultured senescent cells (e.g., human dermal fibroblasts) in a medium containing a predetermined amount (e.g., 5% by volume) of test BRMs for a predetermined time (e.g., 72 hours), and determining that β-Gal activity (e.g., fluorescence intensity measured using a β-Gal measurement kit) is reduced compared to a control condition without BRMs (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); or • Incubating human cultured senescent cells (e.g., human dermal fibroblasts) in a medium containing a predetermined amount (e.g., 5% by volume) of test BRMs for a predetermined time (e.g., 48 hours), and determining that cell proliferation activity (which can be evaluated by a WST-8 assay, etc.) is increased compared to a control condition without BRMs (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 medium containing a predetermined amount (e.g., 5% by volume) of test BRMs, 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 decrease of 0.9 times or less, 0.7 times or less, or 0.5 times or less) compared to a control condition without BRMs. - When highly aged human cultured cells (e.g., P40 or P41 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 test BRMs, the cell number increases (e.g., an increase of at least 5% or at least 10%).BRMs 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 test BRMs, mitochondrial activity (which can be evaluated by ATP assays and NAD / NADH assays, etc.) increases compared to a control condition without BRMs (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 test BRMs, DNA damage (which can be evaluated by γH2AX expression levels, etc.) decreases compared to a control condition without BRMs (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 measured indicators (β-Gal activity, proliferative capacity, etc.) compared to human cultured cells derived from the same type of tissue. Control conditions (such as conditions without BRMs) may be replaced with specified values ​​(specified values ​​can be set by repeated measurements under the same test conditions).

[0044] In one embodiment, the above tests can be performed on the cell culture supernatant, and the BRMs of this disclosure can be prepared using the culture supernatant that is deemed acceptable.

[0045] (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.

[0046] (Example 1: Extracellular Vesicles (EVs) in BRMs) 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 designated as BRMs. As shown below, the BRMs showed excellent effects, so the EVs contained in them were analyzed. In the following examples, unless otherwise specified, BRMs refers to the entire culture supernatant from which extracellular vesicles have not been separated, and BRMs-S refers to the supernatant from which extracellular vesicles have been removed.

[0047] - 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 BRMs. 30 mL of BRMs was further centrifuged at 4°C, 10,000G for 40 minutes for the first centrifugation. 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 in phosphate-buffered saline (PBS) (600 μL) and this was used as 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 for the second centrifugation. 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 "BRMs-S". A Himac CP80NX with a P65A rotor was used for centrifugation at 100,000 G. The presence of extracellular vesicles in the BRMs was confirmed by the use of CD63 and CD81 markers.

[0048] 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.

[0049] The results are shown in Figure 2. BRMs contain approximately 6.15 × 10 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.

[0050] (Example 2: Relationship between BRMs and EV and mTOR and MAPK pathway activity) Based on the results of the study in this example, it was predicted that BRMs 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, we investigated the effects of BRMs and EV on these molecules. Typically, the experiment was conducted according to the following procedure.

[0051] 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.

[0052] Western blot cells were seeded in a 6-well plate. Two days after seeding, BRMs were added to a final concentration of 5%, and sEVs and m / LEVs were added in amounts equivalent to 5% BRMs. The plates were then set at 37°C and 5% CO2. ２ The cells were incubated under the following conditions for 8 hours. Torin-2 was added to the cells along with BRMs or EVs to a final concentration of 250 nmol / L, and incubated at 37°C in 5% CO2. ２ The 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.

[0053] 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.

[0054] 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.

[0055] Antibodies used: Primary antibodies: ATP5A mitochondrial marker (Abacm #ab14848), LC3 (MBL #PM036), p62 (SQSTM1) (MBL #PM045), GAPDH (14C10) (Cell Signaling Technology #2118), ULK-1 (D8H5) (Cell Signaling Technology #8054T), Phospho-ULK1 (Ser757) (Cell Signaling Technology #6888T), p70, S6 kinase (49D7) (Cell Signaling Technology #2708T), Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signaling Technology #9234T) mTOR (7C10) (Cell Signaling Technology #2983T) Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology #5536T) BiP antibody (Cell Signaling Technology #3183S) 4E-BP1 (53H11) (Cell Signaling Technology #9644T) Phospho p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling Technology #4370T) Phospho p38 MAPK (Thr180 / Tyr182) (Cell Signaling Technology #9211S) PhosphoMEK1 / 2 (Ser217 / 221) (Cell Signaling Technology #9121S)

[0056] Secondary antibodies Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology #7074S) Anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology #7076S)

[0057] Protein assay: Protein quantification was performed using Pierce® BCA Protein Assay Kits (#23225 Thermo Scientific®). BRMs were 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.

[0058] (Results) SH-SY5Y cells were treated with BRMs and other substances, 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, BRMs-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, the ERK1 / 2 results indicate that, similarly, activation of the MAPK pathway was observed with BRMs-S, while no activation was observed with sEVs. These results suggest that the supernatant of BRMs during centrifugation 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 BRMs, BRMs-S, and sEVs also suggested the above trend (results not shown).

[0059] Furthermore, when EXO-Prep (Cosmo Bio) was used to separate the EV fraction, it was similarly observed that the supernatant of BRMs 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. Inhibitor BafA1 added only 3. BRMs added 4. EXO-Prep extract added

[0060] (Example 3: Relationship between BRMs and biological functions) The mTOR pathway and the MAPK pathway are associated with various biological functions. Using SH-SY5Y cells, we investigated how BRMs affect biological functions.

[0061] (Effects on Autophagy) To investigate the effects of BRMs on autophagy, LC3 punctae and p62 were examined. The results are shown in Figure 8. Treatment with BRMs reduced the amount of cells containing LC3 punctae and the LC3 band in the Western blot, and accumulation of p62, an autophagy substrate, was observed. This suggests that BRMs tend to suppress autophagy.

[0062] (Effects on mitochondria) To investigate the effects of BRMs 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 BRMs resulted in an enhancement of mitochondrial signaling. BRMs tended to improve mitochondrial activity.

[0063] (Effects on cellular stress) To investigate the effects of BRMs under cellular stress conditions, tunicamycin was used to mimic endoplasmic reticulum stress. Tunicamycin suppresses mTORC1 activity by phosphorylating eIF2α and activating AMPK via the PERK pathway. The results are shown in Figure 10. Treatment with BRMs resulted in increased S6K phosphorylation under endoplasmic reticulum stress conditions, suggesting that BRMs have an effect of alleviating endoplasmic reticulum stress.

[0064] (Discussion) The above experiments suggest that the BRMs of Example 1 interfere with the mTOR and MAPK pathways, affecting autophagy, mitochondrial activity, and cellular stress. Furthermore, it was shown that while the supernatant of BRMs activates the mTOR and MAPK pathways, 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 BRMs of this disclosure are expected to be useful in treating or preventing various diseases. In addition, since the effect was observed in the neuronal cell SH-SY5Y, the BRMs 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 example, is known to be a cause of diabetes and neurological diseases.

[0065] (Example 4: Reduction of aging by BRMs) As described above, BRMs showed effects on the mTOR pathway and the MAPK pathway, modulating biological functions. Based on these molecular behaviors, an effect on aging was expected, so the effect of BRMs on aging was investigated.

[0066] (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.

[0067] The measurements in this embodiment were performed as follows.

[0068] (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. ２Incubate 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).

[0069] (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).

[0070] (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.

[0071] (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.

[0072] (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.

[0073] (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.

[0074] To demonstrate the effect of the BRMs in Example 1, OC-01, a human adipose stem cell culture supernatant (hereinafter also referred to as the "comparative product"), was obtained as a control. When small-sized EVs (sEVs) were extracted from the same volume of BRMs in Example 1 and the comparative product, as shown in Example 1, it was found that the BRMs in Example 1 contained more sEVs than the comparative product (Figure 12). When comparing all EVs, the BRMs in Example 1 also contained more EVs than the comparative product. The total EVs of the comparative product were approximately 5.11 x 10⁶. ９ The 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.

[0075] The effects of the BRMs of Example 1 and the comparative product on young and senescent cells were tested. In each test, the comparative product was added at a concentration of 5% of the culture medium, and the BRMs of Example 1 were added at a concentration of 5% or 10% of the culture medium.

[0076] Figures 13-17 show the effects of the BRMs of Example 1 on β-gal activity, cell proliferation, doubling time, and mitochondrial activity in senescent cells. The BRMs of Example 1 were able to dose-dependently reduce the elevated β-gal activity in senescent cells, whereas the comparative product did not show such an effect (Figure 13). When cell proliferation was measured by WST-8 activity, both the BRMs of Example 1 and the comparative product were observed to activate cell proliferation, but the effect of the BRMs of Example 1 was more potent (Figure 14). A similar trend was observed in doubling time, with the BRMs of Example 1 activating senescent cells more potently and shortening the doubling time compared to 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 BRMs of Example 1 showed a more potent improvement in mitochondrial activity than the comparative product, were more potent than 1 mM NMN, and were 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.

[0077] 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 BRMs of Example 1 (added to the culture medium at a concentration of 10%) on DNA damage was evaluated using γH2AX, it was observed that BRMs of Example 1 significantly reduced the increased γH2AX staining spots in aged cells, indicating that they can reduce DNA damage (Figures 19, 20).

[0078] (Example 4A: Reduction of aging by BRMs from which EVs have been removed) Furthermore, similar to Example 2, the effect of BRMs on aging was investigated, including samples treated with the supernatant fraction of BRMs from which EVs have been removed (BRMs-S).

[0079] (Cell culture and BRM addition) When the senescent HDF cells prepared in Example 4 were further subcultured, the cell number hardly increased from P40. Senescent cells that do not increase in number in this way are called highly senescent cells. Senescent HDF cells were cultured at 37°C in fibroblast growth medium (PromoCell® C-23120) containing 2% fetal bovine serum (FCS), 1% penicillin streptomycin, 1 ng / mL human insulin, and 5 mg / mL human fibroblast growth factor (hb-FGF) at 5% CO2. ２ The cells were cultured, and the following were added depending on the conditions: BRMs; BRMs at a final concentration of 5%, BRMs-S; supernatant fraction obtained from an amount of BRMs equivalent to that of 5% BRMs, NMN; β-nicotinamide mononucleotide (NMN) (Sigma-Aldrich N3501) at a final concentration of 1 mmol / L.

[0080] (Measurement of cell count) 13,000 passage 42 (P42) highly aged HDF cells were seeded and cultured for 7 days under various conditions, after which the cell count was measured. On the 7th day of culture, the culture medium was removed from the culture, washed with PBS, and the cells were detached with Trypsin / EDTA solution and collected. The collected cells were collected by centrifugation, suspended in culture medium, and the cell count was measured using a hemocytometer. The total cell count was calculated based on these results.

[0081] (Cell observation and photography) Cells cultured for 7 days under each condition were morphologically observed and photographed using an inverted fluorescence phase-contrast microscope BZ-X710 (KEYENCE). A 10x lens was used.

[0082] (Measurement of cell proliferation by WST-8 assay) 3 × 10⁶ aged HDF cells at passage number 35 (P35) ３ Seed at a cell / well concentration, at 37°C and 5% CO2. ２ The cells were incubated for 24 hours, then the culture medium was changed to one of the specified conditions, and after incubation for 48 hours, measurements were taken using Cell Counting Kit-8 (Dongrentang). After incubation, 10 μL of WST-8 was added to each well, and the cells were incubated at 37°C and 5% CO2. ２ The samples were incubated for 2.5 hours, and the absorbance at 450 nm was measured using a plate reader (BioTek Synergy 4). Three samples were measured for each condition, and two samples were measured for the Blank (unseeded cells). The measured absorbance value of each sample was subtracted from the average absorbance value of the Blank to obtain the measured value, which was then used for analysis.

[0083] (Results) Figure 21 shows the results of cell count measurement. In Control, where no additives were added, the number of highly aged P42 cells did not increase even after culture, whereas a significant increase in cell count was observed in BRMs and BRMs-S. Figure 22 shows the results of cell proliferation measurement by WST-8 assay. Compared with Control, where no additives were added, the measured values ​​were slightly lower under the NMN-added condition. On the other hand, higher WST-8 activity was shown in both BRMs and BRMs-S conditions than in Control.

[0084] (Discussion) A remarkable effect of promoting the proliferation of senescent cells was confirmed in both stem cell culture supernatants (BRMs) and the supernatant fraction from which EVs were removed. Considering the results of Examples 2 and 3, the above results suggest that the BRMs of this disclosure, including sEVs, 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 both BRMs containing EVs and BRMs without EVs showed superior anti-aging effects compared to NMN, which is known to have anti-aging effects. Furthermore, while anti-aging effects that suppress the acceleration of aging have been reported for various drugs, the effect of making highly senescent cells that have lost their cell proliferation ability to proliferate again has not been reported. Based on the observed anti-aging effects, the BRMs of this disclosure may be applied to the following diseases.

[0085] 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 BRMs of this disclosure may be used in the prevention or treatment of Alzheimer's disease and Parkinson's disease.

[0086] 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 BRMs of this disclosure may be used to prevent or treat diabetes (particularly type 2 diabetes).

[0087] 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 BRMs of this disclosure may be used to prevent or treat cardiovascular diseases (atherosclerosis, hypertension, myocardial infarction, and stroke) by reducing cellular senescence.

[0088] It has been reported that senescent cells accumulate in the lungs due to smoking or air pollution, and that this contributes 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 BRMs of this disclosure may also be used to prevent or treat COPD.

[0089] Senescent cells present in bone or joints have been reported to secrete inflammatory cytokines and promote inflammation. Therefore, the BRMs of this disclosure 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.).

[0090] (Example 5: Cell culture in the presence of BRMs) Furthermore, the effects of BRMs on the subculturing of senescent cells were investigated.

[0091] Senescent cells at passage 38 (P38) in Example 4 were prepared, and the change in cell number when cultured from there to P41 was observed under conditions where 5% BRMs or DMSO (Control) was added to the culture medium. During passage, all cells were collected regardless of the cell proliferation rate, and all collected cells were seeded in a fresh dish. Passing was performed every two weeks.

[0092] The following table shows the cell count results at the end of each subculturing.

[0093] Enhanced cell proliferation was observed with the addition of BRMs. Although senescent cells have reduced proliferative capacity, activation of cell proliferation was observed even in such cells with BRMs, suggesting that even cells with low proliferative capacity may be easier to culture with the addition of BRMs. In particular, P40 and P41 cells, which are considered highly senescent cells as they show loss of proliferative capacity under control conditions, were observed to still be able to proliferate under BRM-added conditions. Cells, by their very nature, possess homeostasis, maintaining their function while undergoing metabolism, so BRMs may be useful in maintaining the health and normal function of cultured cells.

[0094] (Example 6: Effects of BRMs in humans) Since the effects were confirmed at the cellular level in the above examples, the effects of BRMs in humans were tested.

[0095] (Reduction of blood glucose levels) Three subjects (all men in their 40s) in a fasting state were intravenously administered 30 mL of BRMs. Blood samples were taken from each subject before administration of BRMs and 30 minutes after administration. Blood glucose levels were measured from the blood samples.

[0096] The results are shown in the table below. Subjects A1 and A2 represent the results of blood glucose measurements taken before and after different administrations in the same subject. The interval between administrations for A1 and A2 was approximately two months.

[0097] BRMs were observed to reduce the subjects' blood glucose levels within 30 minutes of intravenous administration. Another cell-level study found that BRMs promote insulin release. Furthermore, as mentioned above, BRMs may also improve insulin resistance (sensitivity). It is believed that BRMs can treat diabetes through a mechanism similar to the effects observed at the cell level in the above examples. Similarly, BRMs with the extravasation factor (EV) removed (BRMs-S) are also thought to be able to treat diabetes.

[0098] (Improvement of Chronic Obstructive Pulmonary Disease (COPD)) Male patients exhibiting symptoms of COPD were intravenously administered 30 mL of BRMs every two weeks. Blood samples were taken after each BRM administration. KL-6 levels were measured from the blood samples. KL-6 is a marker of interstitial pneumonia, and a normal value of less than 500 U / mL is generally established; it can also be used to evaluate COPD. Approximately two months before the start of BRM administration, the KL-6 level of the male patient was 748 U / mL.

[0099] The results of the three measurements are shown in the table below.

[0100] At the measurement point after the start of BRMs treatment, KL-6 levels were below the normal range, and improvement in COPD symptoms was observed. Therefore, it was confirmed that BRMs are actually effective in treating COPD in humans. It is thought that a similar mechanism to the effect observed at the cellular level in the above example is at work in the treatment of COPD, and it is believed that not only BRMs but also BRMs-S will produce a similar therapeutic effect.

[0101] This disclosure provides BRMs useful for the treatment of various diseases.

Claims

1. (a) Approximately 6 x 10 １０ (b) BRMs containing small extracellular vesicles (sEVs) of 1 or more cells / mL, or prepared by removing extracellular vesicles (EVs) from the culture supernatant of stem cells.

2. A composition comprising the BRMs described in claim 1 for treating or preventing aging or age-related diseases.

3. A composition for increasing the proliferative capacity of cells, comprising the BRMs described in claim 1.

4. A composition for treating or preventing aging, comprising the BRMs described in claim 1.

5. A composition comprising the BRMs described in claim 1 for enabling the proliferation of highly aged cells.

6. The composition according to claim 2, wherein the aging-related disease includes Alzheimer's disease or Parkinson's disease.

7. The composition according to claim 2, wherein the aging-related disease includes diabetes.

8. The composition according to claim 2, wherein the aging-related disease includes cardiovascular disease.

9. The composition according to claim 2, wherein the aging-related disease includes chronic obstructive pulmonary disease (COPD).

10. The composition according to claim 2, wherein the aging-related disease includes arthritis and bone disease.

11. A composition for modulating mTOR activity or MAPK activity, comprising the BRMs described in claim 1.

12. A composition for use in cell culture, comprising the BRMs described in claim 1.

13. A composition for injection according to any one of claims 2 to 12.

14. The BRMs are approximately 6 × 10 １０ The composition according to any one of claims 2 to 12, wherein the BRMs contain small extracellular vesicles (sEVs) of a certain size per mL or more.

15. Approximately 6×10 １０ BRMs according to claim 1, comprising small extracellular vesicles (sEVs) of 1 or more cells / mL.

16. BRMs according to claim 15, comprising extracellular vesicles (EVs) having a particle size distribution with a mode of approximately 100 to approximately 120 nm.

17. BRMs according to claim 15, wherein the proportion of small-sized extracellular vesicles (sEVs) among all extracellular vesicles is approximately 90% or more.

18. BRMs according to claim 1, prepared by removing extracellular vesicles (EVs) from the culture supernatant of stem cells.

19. BRMs according to claim 1, which are obtained from the culture supernatant of mesenchymal stem cells.

20. The BRMs according to claim 19, 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.

21. A method for preparing a supernatant fraction or an extracellular vesicle fraction of BRMs, comprising the step of separating the BRMs according to any one of claims 1 and 15 to 20 into a supernatant fraction and an extracellular vesicle fraction.

22. A supernatant fraction, an extracellular vesicle fraction, or a combination comprising the supernatant fraction and the extracellular vesicle fraction obtained by the method of claim 21.

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

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

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