Exosome production promoter and exosome production promotion method

Abstract

The present invention provides an exosome production promoter that promotes the production of exosomes from adipose-derived mesenchymal stem cells. The exosome production promoter that promotes the production of exosomes from adipose-derived mesenchymal stem cells cultured in a serum-free medium is characterized by having EGF. Additionally, it is preferable to include IL-1β and trehalose. It is preferable not to include SCGF. It is preferable to culture using a nonwoven sheet as a scaffold. Exosomes mass-produced by the method according to the present invention can be used for purposes such as cosmetic surgery.

Description

Technology Field

[0001] The present invention relates to an exosome production promoter that promotes exosome production from mesenchymal stem cells, and a method for promoting exosome production. Background Technology

[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells found in mesenchymal tissue, that is, connective tissue derived from the mesoderm. It has been revealed that in an undifferentiated state, mesenchymal stem cells secrete cytokines and growth factors that have anti-inflammatory, pro-cell proliferation, and pro-angiogenic effects, and support tissue regeneration through paracrine (Non-patent Literature 1). The molecular groups secreted by undifferentiated mesenchymal stem cells are not limited to specific diseases but have therapeutic effects on a variety of diseases. Tissue regeneration becomes possible by using sensitive mesenchymal stem cells without inducing differentiation into the tissue cells to be regenerated, and furthermore, without applying any artificial manipulation such as genetic modification to the cells.

[0003] Exosomes can mediate intercellular information transfer by transporting proteins, lipids, and RNA between cells (Non-patent Literature 2, 3). It has been found that molecules contained in exosomes, such as proteins, microRNA, and mRNA, possess functions similar to those of the cells from which they originate. Therefore, exosomes secreted by mesenchymal stem cells, which are attracting attention as raw materials for cell therapy for a wide range of diseases, are expected to possess therapeutic effects similar to those of MSCs (Non-patent Literature 4).

[0004] Patent Document 1 describes exosomes isolated from cultured neural stem cell lines (NSCL). These exosomes can promote the migration of fibroblasts, the branching of human umbilical vein endothelial cells, and the elongation of neurites.

[0005] Exosomes contain relatively less animal serum compared to stem cells, so the risk of symptoms caused by animal serum infection can also be eliminated. Therefore, cell therapy using exosomes can overcome the limitations of existing stem cell therapies.

[0006] Patent Document 2 discloses an exosome production promoter having a sphingoid base as an active ingredient. However, the exosome production promoter described in Patent Document 2 is limited to those capable of promoting exosome production in nerve cells to cure Alzheimer's disease. Prior art literature

[0007] Patent Document 1: International Publication No. 2013 / 150303 Patent Document 2: Japanese Patent Publication No. 2019-156786

[0008] Non-patent Document 1: Chamberlain G, Fox J, Ashton B, Middleton J, Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25 (2007) 2739-2749 Non-patent document 2: Simons and Raposo, Curropin Cell Biology 2009;21:575-581 Non-patent document 3: Skog et al., Nat Cell Biology 2008;10:1470-1476; Valadi et al., Nat Cell Biology 2007;9:654~659Non-patent document 4: Katsuda T, Kosaka N, Takeshita F, Ochiya T. The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. Proteomics. 13 (2013) 1637-1653. The problem to be solved

[0009] The present invention was made in consideration of these problems and aims to provide an exosome production promoter and a method for promoting exosome production that promotes exosome production from adipose-derived mesenchymal stem cells. means of solving the problem

[0010] The exosome production promoter according to the present invention is characterized by having EGF as an exosome production promoter that promotes exosome production from adipose-derived mesenchymal stem cells cultured in a serum-free medium.

[0011] The exosome production promotion method according to the present invention is a method for promoting exosome production from adipose-derived mesenchymal stem cells, characterized by culturing adipose-derived mesenchymal stem cells in a serum-free medium containing EGF. Effects of the invention

[0012] According to the present invention, the production of exosomes from adipose-derived mesenchymal stem cells can be promoted. Brief explanation of the drawing

[0013] Figure 1 is a diagram showing the production of exosomes from adipose-derived mesenchymal stem cells when various factors are added. Specific details for implementing the invention

[0014] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. These embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which the configurations of the following embodiments are appropriately substituted by a person skilled in the art are also included within the scope of the present invention.

[0015] The exosome production promoter according to the present invention promotes the production of exosomes from adipose-derived mesenchymal stem cells cultured in a serum-free medium.

[0016] Adipose-derived mesenchymal stem cells are preferably human adipose-derived mesenchymal stem cells. Compared to bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells possess advantageous characteristics such as rapid cell proliferation, high secretion of regenerative-promoting factors, and high immunosuppressive capacity. Furthermore, since adipose-derived mesenchymal stem cells are obtained from adipose tissue in the abdomen or buttocks, they also possess the advantageous characteristic of being easy to secure in sufficient quantities safely compared to bone marrow-derived mesenchymal stem cells, which require the harvesting of bone marrow. Additionally, using mesenchymal stem cells derived from a patient's own adipose tissue for patient treatment is superior in that it is free from ethical issues, causes no immune rejection, has fewer problems such as infections, and is effective through intravenous administration.

[0017] Adipose-derived mesenchymal stem cells are positive for, for example, CD73, CD90, CD105, and CD44, and negative for CD11b, CD19, CD34, CD45, and HLA-DR. Adipose tissue-derived mesenchymal stem cells have the ability to differentiate into, for example, osteoblasts, adipocytes, and chondrocytes.

[0018] Mesenchymal stem cells derived from adipose tissue are obtained by isolating mesenchymal stem cells from adipose tissue, primary culturing the mesenchymal stem cells in a serum-containing medium, and proliferating the mesenchymal stem cells subcultured from the primary culture in a serum-free medium. Adipose tissue is obtained by surgical resection from mammals, such as humans. Local anesthesia may be used during surgical resection. Alternatively, adipose tissue may be obtained by aspiration by inserting a catheter into the subcutaneous adipose tissue of the abdomen, thigh, or buttocks. The amount of adipose tissue obtained is, for example, 1 g to 100 g, 2 g to 50 g, 2 g to 40 g, or 2 g to 20 g, but is not limited thereto.

[0019] The obtained adipose tissue is washed, for example, with physiological saline and dispersed by immersion at 37°C for 70 minutes in a solution containing collagenase diluted to a concentration of 0.1% in Hanks' equilibrium salt solution. The adipose tissue-derived cells dispersed from the adipose tissue are centrifuged at 1800 rpm for 10 minutes. Additionally, the stromal vascular cell population obtained by centrifugation is diluted with a solution and filtered through a 100 µm nylon mesh. The filtered cells are washed with MEMα.

[0020] Serum-free media include serum-free media for mesenchymal stem cells, such as DMEM, MEMα, DMEM / F12, and MEM.

[0021] The exosome production promoter according to the present invention has EGF. As disclosed in the examples described below, the amount of exosomes produced from adipose-derived mesenchymal stem cells containing a plurality of factors is greater than the amount of exosomes produced from adipose-derived mesenchymal stem cells containing a factor from which only EGF has been removed from these plurality of factors. The inventors have discovered through a new discovery that EGF is a factor that helps in the production of exosomes, and have completed the present invention based on this fact.

[0022] EGF is a protein of 6045 Da composed of 53 amino acid residues and 3 intramolecular disulfide bonds. It binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface and plays an important role in regulating cell growth and proliferation. The EGF content in the medium is not particularly limited, but is preferably 0.05 to 300 ng / ml as the final concentration, and more preferably 1 to 100 ng / ml.

[0023] In addition, it is desirable to include IL-1β to promote the production of exosomes from adipose-derived mesenchymal stem cells. The IL-1β content in the medium is not particularly limited, but is preferably 0.001 to 10 ng / ml at the final concentration, and more preferably 0.01 to 1 ng / ml.

[0024] In addition, it is desirable to include trehalose to promote exosome production from adipose-derived mesenchymal stem cells. Trehalose is a disaccharide formed by 1,1-glycosidic linkages between two α-glucose molecules. The trehalose content in the medium is not particularly limited, but is preferably 1 to 50 mg / ml at the final concentration, and more preferably 10 to 30 mg / ml.

[0025] It is preferable that the exosome production promoter according to the present invention does not contain SCGF. As disclosed in the examples described below, the exosome production from adipose-derived mesenchymal stem cells containing a plurality of factors is less than the exosome production from adipose-derived mesenchymal stem cells containing a factor from which only SCGF has been removed from these plurality of factors. The inventors have discovered through novel findings that SCGF is a factor that inhibits the production of exosomes. Therefore, it is preferable that the exosome production promoter according to the present invention does not contain SCGF. SCGF is a 29-kDa protein that does not have an N-type sugar chain and belongs to the C-type lectin family.

[0026] In addition, since TNFα also inhibits the production of exosomes from adipose-derived mesenchymal stem cells, it is preferable that the exosome production promoter according to the present invention does not contain TNFα.

[0027] In the present invention, it is preferable that adipose-derived mesenchymal stem cells be cultured using a nonwoven fabric scaffold. When adipose-derived mesenchymal stem cells are cultured using a nonwoven fabric scaffold, the production of exosomes is further promoted.

[0028] The weight of the nonwoven fabric should be 1 to 500 g / m², and for example, 50 to 200 g / m² is preferred. The nonwoven fabric may be a hydrophilized nonwoven fabric. Hydrophilization of the nonwoven fabric can be carried out by fluorine gas treatment, atmospheric pressure plasma treatment, vacuum plasma treatment, corona treatment, graft polymerization of hydrophilic monomers, sulfonation treatment, or surfactant application treatment, and fluorine gas treatment and atmospheric pressure plasma treatment are preferred.

[0029] It is preferable that the fibers constituting the nonwoven fabric be polyolefin polymers, and examples of polyolefin polymers include ethylene, propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 1-hexene, 1-octene, 1-dodecene, 1-octadecene, etc.

[0030] The fibers constituting the nonwoven fabric are preferably fibers with a small fiber diameter, and the average fiber diameter is preferably 200 μm or less, more preferably 10 to 100 μm, and particularly preferably 15 to 50 μm. The fibers may include a mixture of fibers with a relatively large fiber diameter and fibers with a relatively small fiber diameter.

[0031] It is desirable for the nonwoven fabric to have porous properties. Porous properties are important in that they supply sufficient oxygen and nutrients to cells necessary for tissue regeneration and rapidly expel carbon dioxide or waste products. In addition, having porous properties increases the specific surface area, thereby increasing cell adhesion. In such cases, the average pore diameter is, for example, 5㎛ to 200㎛, 20㎛ to 100㎛, 25㎛ to 100㎛, 30㎛ to 100㎛, 35㎛ to 100㎛, 40㎛ to 100㎛, 50㎛ to 100㎛, or 60㎛ to 100㎛, and preferably 50㎛ to 300㎛.

[0032] The form of the nonwoven fabric is not particularly limited, but is preferably a nonwoven sheet. The nonwoven sheet preferably has a plurality of through holes penetrated in the thickness direction. The total film thickness of the nonwoven sheet may, for example, be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, or 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. Preferably, it is 30 to 2000 μm, and more preferably, 500 to 1000 μm.

[0033] Exosomes produced by using the exosome production promoter according to the present invention can be used as exosome-containing preparations. The form of the exosome-containing preparation is not particularly limited, but may be a liquid preparation such as an injection, suspension, solution, or spray, for example, or may be a sheet-type preparation or a gel-type preparation.

[0034] Exosome-containing preparations are not limited in their method of use and, for example, can be used to administer directly to a subject or as a source for tissue or organ reconstruction performed in vitro.

[0035] Exosome-containing preparations can be used to treat diseases or disorders. Diseases or disorders that may be targeted include immune diseases, ischemic diseases (lower limb ischemia, ischemic heart disease (myocardial infarction, etc.), coronary heart disease, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, etc.), neurological diseases, Crohn's disease, graft-versus-host disease (GVHD), inflammatory bowel disease including ulcerative colitis, collagen diseases including systemic lupus erythematosus, liver cirrhosis, cerebral infarction, intracerebral hematoma, cerebral vasospasm, radiation enteritis, atopic dermatitis, multiple sclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus, diabetes mellitus, mycosis fungoides (Alibert-Bazin syndrome), scleroderma, diseases arising from degeneration and / or inflammation of connective tissues such as cartilage, eye diseases, neovascularization-related diseases, congestive heart failure, cardiomyopathy, wounds, epithelial damage, fibrosis, lung diseases, cancer, etc.

[0036] In addition, exosome-containing preparations can also be used for cosmetic treatment, treatment, or improvement. Cosmetic purposes include not only treatments for the purpose of improving health conditions purely for aesthetic purposes, but also cosmetic treatments for deformities after surgery or trauma and congenital deformities. For example, they can be used for breast augmentation (breast augmentation, breast reconstruction), tissue augmentation for sunken cheeks or upper and lower eyelids, and tissue augmentation for Perry-Romberg syndrome, facial or funnel chest.

[0037] Exosome-containing preparations may contain pharmaceutically acceptable carriers or additives in addition to exosomes. Such carriers or additives may include, but are not limited to, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, disinfectants or antimicrobial agents, pH adjusters, stabilizers, chelating agents, oily bases, gelling agents, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizing agents, antioxidants, sweeteners, acidifiers, coloring agents, flavoring agents, fragrances, or refreshing agents.

[0038] According to the present invention, it becomes possible to stably supply large quantities of exosomes from adipose-derived mesenchymal stem cells. By using genetic modification technology to overexpress molecules with therapeutic effects, such as mRNA, miRNA, and proteins, in mesenchymal stem cells and producing exosomes loaded with these molecules, it becomes possible to apply them to various treatments and cosmetic applications. Furthermore, by modifying the surface of exosomes, such as by using genetic modification technology to express receptors for proteins that are specifically expressed in target tissues on the surface of the exosomes, it is also possible to deliver exosomes more specifically to target tissues.

[0039] Examples

[0040] Adipose-derived mesenchymal stem cells (ADSC) 4x10 4 The cells were sown in each well. For cell culture, a non-adhesive 24-well plate (PrimeSurface (registered trademark) Sumitomo Bakelite) was used. The basal medium was 1.5 ml of 10% FBS / DMEM F12 (SIGMA). The culture period was 6 days. Starting from Day 2, the medium was changed daily by 1.5 ml, and the sugar consumption of the used culture medium was measured.

[0041] Next, for the 48-hour cell culture on days 7 and 8, DMEM F12 was used as the medium to perform exosome recovery. During this 48-hour culture, 10% ExoFBS (FUNAKOSHI) was added in Test #1. ExoFBS is a form from which bovine exosomes have been removed, and its effect on exosome research is suppressed. Additionally, during the 48-hour culture, all eight factors A through H were added in Test #2 at the concentrations shown in Table 1 below.

[0042]

[0043] In the 48-hour culture, in Test #3, only Factor A was removed from the 8 factors shown in Table 1 and the rest were added; in Test #4, only Factor B was removed from the 8 factors shown in Table 1 and the rest were added; in Test #5, only Factor C was removed from the 8 factors shown in Table 1 and the rest were added; in Test #6, only Factor D was removed from the 8 factors shown in Table 1 and the rest were added; in Test #7, only Factor E was removed from the 8 factors shown in Table 1 and the rest were added; in Test #8, only Factor F was removed from the 8 factors shown in Table 1 and the rest were added; in Test #9, only Factor G was removed from the 8 factors shown in Table 1 and the rest were added; and in Test #10, only Factor H was removed from the 8 factors shown in Table 1 and the rest were added.

[0044] The amount of exosomes produced from ADSCs was measured using an Exo Counter (KENWOOD) with 50 μl aliquots from 1.5 ml of the recovered culture supernatant. The Exo Counter detects exosomes using a sandwich ELISA with a disk and nano-bead antibody to specifically detect surface antigens. The amount of exosomes was quantified by detecting CD81 on the exosome surface.

[0045] The results are shown in Figure 1. As shown in Figure 1, when EGF was removed, the amount of exosomes produced was reduced, and it was found that EGF is a factor that helps produce exosomes. In addition, when IL-1β was removed, the amount of exosomes produced was reduced, and it was found that IL-1β is also a factor that helps produce exosomes. In addition, when trehalose was removed, the amount of exosomes produced was reduced, and it was found that trehalose is also a factor that helps produce exosomes.

[0046] Conversely, when SCGF was removed, the amount of exosomes produced increased, proving that SCGF is a factor that inhibits exosome production. Additionally, when TNFα was removed, the amount of exosomes produced increased, proving that TNFα is also a factor that inhibits exosome production. Industrial applicability

[0047] It can be used for the production of exosomes.

Claims

Claim 1 An exosome production promoter that increases the number of exosomes produced from adipose-derived mesenchymal stem cells (ADSCs) cultured in a serum-free medium, wherein the exosomes are CD81 positive and the exosome production promoter has Epidermal Growth Factor (EGF). Claim 2 An exosome production promoter according to claim 1, characterized by additionally including IL-1β. Claim 3 An exosome production promoter according to claim 1 or 2, characterized by additionally including trehalose. Claim 4 An exosome production promoter characterized by not including SCGF (Stem Cell Growth Factor) in claim 1 or 2. Claim 5 An exosome production promoter according to claim 1 or 2, characterized by not containing TNFα. Claim 6 An exosome production promoter according to claim 1 or 2, characterized in that the adipose-derived mesenchymal stem cells are cultured using a nonwoven fabric scaffold. Claim 7 A method for promoting exosome production to increase the number of exosomes produced from adipose-derived mesenchymal stem cells, wherein the exosomes are CD81 positive, and the method for promoting exosome production is characterized by culturing adipose-derived mesenchymal stem cells in a serum-free medium containing EGF. Claim 8 A method for promoting exosome production according to claim 7, characterized in that the serum-free medium further comprises IL-1β. Claim 9 A method for promoting exosome production according to claim 7 or 8, characterized in that the serum-free medium further comprises trehalose. Claim 10 A method for promoting exosome production according to claim 7 or 8, characterized in that the exosome production promoting method does not include SCGF in the serum-free medium. Claim 11 A method for promoting exosome production according to claim 7 or 8, characterized in that the serum-free medium does not contain TNFα. Claim 12 A method for promoting exosome production according to claim 7 or 8, characterized in that the adipose-derived mesenchymal stem cells are cultured using a nonwoven fabric scaffold.

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