Stem cell cultured supernatant liquid, and method for producing the same

Abstract

To provide stem cell cultured supernatant liquid having activity different from conventional liquid.SOLUTION: It has been found out that stem cell cultured supernatant liquid having excellent activity was obtained by co-culturing a mesenchymal stem cell derived from two kinds or more of different tissues. The present disclosure relates to a method for producing mesenchymal stem cell cultured supernatant liquid including: i) co-culturing two kinds or more of mesenchymal stem cells; and ii) recovering cultured supernatant liquid. Cell cultured supernatant liquid obtained by the method is applicable to medicines or cosmetics.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a stem cell culture supernatant and a method for producing the same, more specifically, to a mesenchymal stem cell culture supernatant produced by co-culturing mesenchymal stem cells derived from multiple tissues and a method for producing the same, and further relates to a pharmaceutical composition or cosmetic composition containing the mesenchymal stem cell culture supernatant or a dry powder thereof. [Background technology]

[0002] Mesenchymal stem cells are somatic stem cells present in bone marrow, synovium, fat, and umbilical cord, and have been reported to have the ability to differentiate into cartilage, bone, fat, and nerve cells (Non-Patent Document 1). Because they can be isolated from adult tissues, they are used in regenerative treatment of damaged areas of the meniscus and cartilage (Non-Patent Document 2). In addition, mesenchymal stem cells also have anti-inflammatory and immunoregulatory effects, and are therefore known to have been widely used for various diseases, such as liver disease, graft-versus-host disease, and autoimmune diseases.

[0003] In this context, it has become clear that the function of these mesenchymal stem cells transplanted into the body in cell transplantation therapy is greatly influenced by physiologically active substances such as various cytokines secreted by mesenchymal cells.

[0004] When mesenchymal stem cells are cultured in vitro, these physiologically active substances are released into the culture medium. There have been reports of successful cases in which the culture medium used for culturing mesenchymal stem cells was collected and used to regenerate tissues, since the culture medium contains a large amount of substances released from the cells (Non-Patent Documents 3, 4, and 5).

[0005] In this way, the culture supernatant produced during the culture process of mesenchymal stem cells is rich in signaling substances that play an important role in cell activity, such as growth factors and immune regulatory factors (cytokines) secreted from stem cells, including exosomes, and is thought to be useful for restoring the functions of damaged tissues and cells in the body. The exosomes, growth factors, and cytokines abundant in the supernatant act on surrounding stem cells, and are expected to have a functional recovery effect such as tissue regeneration and improved immunity by collecting cells at areas where function has been reduced due to aging or damage. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Colter, DC, Sekiya, I. & Prockop, DJ Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci. 2001;98:7841-7845. [Non-Patent Document 2] Sekiya I., Muneta T., Horie M. & Koga H. Arthroscopic Transplantation of Synovial Stem Cells Improves Clinical Outcomes in Knees With Cartilage Defects. Clin Orthop Relat Res. 2015;473:2316-26. [Non-Patent Document 3] Osugi M., Katagiri, W., Yoshimi, R., Inukai, T., Hibi, H., Ueda M. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects. Tissue Engineering Part A.2012;18:1479-1489 [Non-Patent Document 4] Shohara, R., Yamamoto, A., Takikawa, S., Iwase, A., Hibi, H., Kikkawa, F., Ueda, M. Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms. Cytotherapy,2012;14(10):1171-1181 [Non-Patent Document 5] Kawai, T., Katagiri, W., Osugi, M., Sugimura, Y., Hibi, H., Ueda, M. Secretomes from bone marrow-derived mesenchymal stromal cells enhance periodontal tissue regeneration. Cytotherapy,2015;17(4):369-381 Summary of the Invention [Problem to be solved by the invention]

[0007] One of the objectives of the present disclosure is to provide a stem cell culture supernatant having an activity different from that of conventional products. Another objective of the present disclosure is to provide a pharmaceutical and / or cosmetic product based on a stem cell culture supernatant having an activity different from that of conventional products. [Means for solving the problem]

[0008] The present inventors have found that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant containing unexpected components and having excellent activity can be obtained.

[0009] The present disclosure is based on these findings and includes the following aspects: (Embodiment 1) A method for producing a mesenchymal stem cell culture supernatant, comprising: i) co-culturing two or more types of mesenchymal stem cells; and ii) Recovering the culture supernatant A method comprising: (Embodiment 2) The method of embodiment 1, further comprising separately culturing said two or more types of mesenchymal stem cells prior to co-culturing. (Embodiment 3) The method of embodiment 1, wherein the mesenchymal stem cells are cells derived from a tissue selected from the group consisting of adipose, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, and hair follicles. (Embodiment 4) The method of embodiment 1, wherein two types of mesenchymal stem cells are used. (Embodiment 5) The method of embodiment 1, wherein human adipose stem cells and human umbilical cord stem cells, human adipose stem cells and human amniotic stem cells, or human umbilical cord stem cells and human amniotic stem cells are used. (Embodiment 6) The method according to embodiment 1, wherein the mesenchymal stem cell culture supernatant contains a component that is not present when the two or more types of mesenchymal stem cells are cultured individually. (Embodiment 7) The method of embodiment 1, wherein the mesenchymal stem cell culture supernatant contains a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), Glutamate-cysteine ​​ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3). (Embodiment 8) The method of embodiment 1, wherein the mesenchymal stem cell culture supernatant contains an increased amount of a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), Glutamate-cysteine ​​ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3) compared to the amount contained when the two or more types of mesenchymal stem cells are cultured individually. (Embodiment 9) The method of embodiment 1, comprising culturing the cells in a serum-free medium that maintains the undifferentiated state of MSCs, a medium containing serum added to a basal medium, a culture medium for human mesenchymal stem cells, a feederless medium for mesenchymal stem cells, and / or a chemically defined serum-free medium. (Embodiment 10) A cell culture supernatant obtained by the method according to any one of embodiments 1 to 9. (Embodiment 11) A pharmaceutical composition comprising a cell culture supernatant obtained by the method according to any one of embodiments 1 to 9. (Embodiment 12) A cosmetic composition comprising a cell culture supernatant obtained by the method according to any one of embodiments 1 to 9. (Embodiment 13) A method for producing a mesenchymal stem cell culture supernatant powder, comprising freeze-drying a cell culture supernatant obtained by the method according to any one of embodiments 1 to 9. (Embodiment 14) Mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13. (Embodiment 15) A pharmaceutical composition comprising mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13. (Embodiment 16) A cosmetic composition comprising mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13. (Embodiment 17) 12. The pharmaceutical composition of embodiment 11 for use in the treatment, prevention or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases. (Embodiment 18) 16. The pharmaceutical composition of embodiment 15 for use in the treatment, prevention or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 is a schematic diagram showing a production flow of a culture supernatant. [Figure 2A] 13 is a photograph of cells at the time of extracting culture supernatant from a co-culture of human adipose-derived MSCs and human umbilical cord-derived MSCs. [Figure 2B] 13 is a photograph of cells at the time of extracting culture supernatant from a co-culture of human adipose-derived MSCs and human amnion-derived MSCs. [Figure 2C] 13 is a photograph of cells at the time of extracting culture supernatant from a co-culture of human umbilical cord-derived MSCs and human amnion-derived MSCs. [Figure 3A] 1 is a graph showing the results of proteomic analysis of RND3 shown in Table 1. [Figure 3B] 1 is a graph showing the results of proteomic analysis of GCLC shown in Table 1. [Figure 3C] 1 is a graph showing the results of proteomic analysis of RAD21 shown in Table 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] As described above, the present inventors have found that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant having unexpectedly excellent activity can be obtained. The present invention will be described in detail below.

[0012] (Co-culture of MSCs) One embodiment of the present disclosure relates to a method for producing a mesenchymal stem cell culture supernatant, more specifically, the method includes, for example, i) co-culturing two or more types of mesenchymal stem cells, and ii) recovering the culture supernatant. Another embodiment of the present disclosure relates to a cell culture supernatant obtained by the above-mentioned production method.

[0013] Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in multiple tissues, including the umbilical cord, bone marrow, and adipose tissue. Although MSCs are rare cells, they are estimated to be present in bone marrow at a ratio of 1 per 10,000 to 100,000 nucleated bone marrow cells. MSCs have the ability to self-renew and can differentiate into various cell types, including osteoblasts (bone cells), chondrocytes, muscle cells, and adipocytes.

[0014] In some embodiments, mesenchymal stem cells used in the methods of the present disclosure include, but are not limited to, cells isolated from human tissues, such as adipose, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, Cord Placenta Junction (CPJ), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, or hair follicles.

[0015] According to the definition by Dominici et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement., Cytotherapy. 2006;8(4):315-7, mesenchymal stem cells (MSCs) are (1) cells that adhere to plastic culture vessels, (2) cells with positive markers of CD105 (endoglin), CD73 (ecto-5'-nucleotidase), and CD90 (Thy-1) and negative markers of CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-Class II (DR), and (3) cells that have the ability to differentiate into bone, fat, and cartilage. Conventional methods for isolating mesenchymal stem cells utilize the property of MSCs that they adhere to plastic culture vessels and proliferate, but MSCs can also be isolated based on cell surface markers using FACS (fluorescence activated cell sorting) and the like. In FACS, cells are stained with fluorescent antibodies, and specific cells are sorted by measuring and analyzing the fluorescence emitted by each cell. As the FACS system, for example, a BD FACSAria (trademark) cell sorter manufactured by BD Biosciences can be used.

[0016] As mentioned above, mesenchymal stem cells are often isolated by culturing cells obtained from tissues such as bone marrow for a long period of time and then proliferating the cells that adhere to the culture dish. However, techniques have also been developed to isolate mesenchymal stem cells using surface antigen markers. For example, Ogata et al. have demonstrated that human MSCs can be purified using two cell surface markers, LNGFR and THY-1 (Ogata et al., PLoS One. 2015 Jun 8;10(6):e0129096.).

[0017] To be identified as MSCs, greater than 95% of the cultured cell population must express CD73, CD90 and CD105, and be negative (≦2% positive cells) for CD11b or CD14, CD34, CD45, CD19 or CD79α, and HLA-DR.

[0018] In some embodiments, the method according to the present disclosure includes a step of co-culturing two or more kinds of mesenchymal stem cells. For example, co-culture can be performed with a combination of any two or more kinds (e.g., two, three, four, five or more kinds) of MSCs derived from fat, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, or hair follicle. For example, two or all three kinds of adipose stem cells, umbilical cord stem cells, and amniotic stem cells can be selected for co-culture, for example, a combination of human adipose stem cells and human umbilical cord stem cells, a combination of human adipose stem cells and human amniotic stem cells, or a combination of human umbilical cord stem cells and human amniotic stem cells can be used, but is not limited thereto.

[0019] In some embodiments, the methods of the present disclosure may include the step of separately culturing two or more types of mesenchymal stem cells prior to co-culturing.

[0020] For cell culture, adhesion culture, suspension culture, rotation culture, hanging drop culture, nonwoven fabric culture, or tube culture may be used, but is not limited to these.

[0021] For cell culture, a culture vessel such as a culture vessel for adhesion culture, e.g., CellBIND available from Corning, tissue culture dishes available from Corning, cell culture dishes available from Falcon, tissue culture dishes available from IWAKI, CELLSTAR Advanced petri dishes available from Greinar, etc. can be used, but are not limited to these.

[0022] The cell seeding rate is, for example, 1,000 to 25,000 cells / cm. 2 A cell density of, for example, about 1,000 cells / cm 2 , about 2,000 cells / cm 2 , about 3,000 cells / cm 2 , approximately 4,000 cells / cm 2 , approximately 5,000 cells / cm 2 , approximately 6,000 cells / cm 2 , about 7,000 cells / cm 2 , about 8,000 cells / cm 2 , approximately 9,000 cells / cm 2 or approximately 10,000 cells / cm 2 The cell density can be set at 100 to 2000. As used herein, the term "about" means + / - 10% of the stated value. In other words, in the present specification, the expression "about 1,000" can mean any numerical value within the range of 900 to 1,100.

[0023] For cell culture, a culture medium that can be used is, for example, a serum-free medium that maintains the undifferentiated state of MSCs, a medium in which serum has been added to a basal medium, a culture medium for human mesenchymal stem cells, a feederless medium for mesenchymal stem cells, or a chemically defined serum-free medium, such as a culture medium for human mesenchymal stem cells available from Nipro (#87-072), StemFit For Mesenchymal Stem Cell available from Ajinomoto Co., KBM ADSC-4 medium (#16030044) available from Kohjin Bio Co., Ltd., or R:STEM Medium for hMSC High Growth available from Rohto Pharmaceutical Co., Ltd., but is not limited to these.

[0024] Typically, cells are cultured at 37°C and 5% CO2, but conditions can be adjusted accordingly. Cells may be cultured under hypoxic or hyperoxic conditions, and may be cultured in adherent or suspension culture, or in large-scale culture using microbeads or nonwoven fabrics.

[0025] The frequency of medium replacement can be, for example, once a day, once every two days, once every three days, once every four days, once every five days, or once a week.

[0026] When isolating MSCs by adhesion culture, the tissue-derived cells can be passaged at least once. The number of passages for isolating MSCs can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example.

[0027] MSCs derived from two or more different tissues can be cultured separately, and then the MSCs derived from two or more different tissues can be co-cultured. During co-culture, each MSC can be seeded with equal cell numbers (or ratios), or different cell numbers (or ratios). When two types of cells are used, the ratio can be, for example, 50:50, 40:60, 30:70, 20:80, or 10:90, but is not limited thereto. When two types of cells are used, the ratio can be, for example, about 50:50. When three or more types of cells are used, any ratio can be used.

[0028] In some embodiments, the culture supernatant may be collected after the co-cultured cells reach confluence. The culture supernatant refers to the supernatant collected during the cell culture process. The culture supernatant contains tens to hundreds of types of cytokines and growth factors, which are a type of protein, and is expected to have a wide range of therapeutic effects. In addition, the culture supernatant does not contain genetic information (cell components and DNA), and is less likely to cause rejection or foreign body reactions when administered, making it highly versatile. In some embodiments, the culture supernatant may be collected after the co-cultured cells reach confluence, after the medium is replaced, and after further culturing (e.g., after culturing for 12 hours, 1 day, 2 days, 3 days, 4 days, or 5 days).

[0029] In some embodiments, the recovered supernatant may be sterilized (e.g., filter sterilized). In some embodiments, the recovered supernatant may be frozen (e.g., frozen at -80°C). In some embodiments, the recovered supernatant may be lyophilized. In some embodiments, the lyophilized supernatant may be powdered. In some embodiments, the powdered supernatant may be further tableted.

[0030] One aspect of the present disclosure relates to a method for producing a mesenchymal stem cell culture supernatant powder, comprising: i) co-culturing two or more types of mesenchymal stem cells; ii) recovering the culture supernatant; and iii) freeze-drying the recovered culture supernatant. Another aspect of the present disclosure relates to a cell culture supernatant powder obtained by the above-mentioned method.

[0031] In some embodiments, the cell culture supernatant obtained by the co-culture method according to the present disclosure contains components that are not present when two or more types of mesenchymal stem cells are cultured individually. The components contained in the cell culture supernatant can be examined, for example, by proteome analysis. Table 1 lists components that are contained in the cell culture supernatant obtained by the co-culture method according to the present disclosure but are not present when each mesenchymal stem cell is cultured individually.

[0032] In some embodiments, the mesenchymal stem cell culture supernatant contains a component selected from the group consisting of Glutamate-cysteine ​​ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21). These components are not present in the cell culture supernatant when adipose-, umbilical cord-, or amniotic membrane-derived MSCs are cultured individually, but are present in the cell culture supernatant when two of adipose MSCs, umbilical cord MSCs, or amniotic membrane MSCs are co-cultured in combination.

[0033] Thus, another embodiment of the present disclosure relates to a mesenchymal stem cell culture supernatant liquid containing an ingredient selected from the group consisting of Glutamate-cysteine ​​ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21).Furthermore, another embodiment of the present disclosure relates to a mesenchymal stem cell culture supernatant powder containing an ingredient selected from the group consisting of Glutamate-cysteine ​​ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21).

[0034] Another aspect of the present disclosure relates to a method for producing a protein selected from the group consisting of Glutamate-cysteine ​​ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21).

[0035] In some embodiments, the cell culture supernatant obtained by the co-culture method of the present disclosure contains an increased amount of a component selected from the group consisting of Glutamate-cysteine ​​ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21) compared to the amount contained when two or more types of mesenchymal stem cells are cultured individually.

[0036] Pharmaceutical Composition One embodiment of the present disclosure relates to a pharmaceutical composition comprising the above-mentioned cell culture supernatant liquid or the above-mentioned cell culture supernatant powder.

[0037] In general, MSC culture supernatant is believed to have effects such as regeneration and repair of tissues such as skin, wound healing, and angiogenesis. As shown in Table 2, for example, Glutamate-cysteine ​​ligase catalytic subunit (GCLC) acts suppressively against cancer (e.g., colon cancer), and therefore, the MSC culture supernatant according to the present disclosure containing GCLC can be used as an anticancer drug for the treatment / prevention of cancer. Furthermore, Rho-related GTP-binding protein RhoE (RND3) is necessary for the formation and myelination of oligodendrocytes, and the MSC culture supernatant according to the present disclosure containing RND3 can be used for the treatment / prevention of demyelinating diseases such as multiple sclerosis. Furthermore, double-strand-break repair protein rad21 homolog (RAD21) is known to play an important role in the development of the corneal stroma, and the MSC culture supernatant according to the present disclosure containing RAD21 can be useful as a drug for eye diseases and eye disorders (e.g., including scleralized cornea). Furthermore, all of these factors are involved in cell death and injury caused by UV stimulation, and may be useful in cosmetics (such as sunscreen creams).

[0038] Some embodiments relate to a pharmaceutical composition for use in the treatment, prevention, or amelioration of a disease or disorder selected from the group consisting of cancer, a demyelinating disease, an ocular disease, and an ocular disorder.

[0039] The pharmaceutical composition according to the present disclosure may take any form, such as a tablet, powder, liquid, or semisolid. The pharmaceutical composition according to the present disclosure may be an external medicine, such as a liniment for topical treatment applied through the skin or directly to a skin lesion, and may be prepared by blending various main agents with a base. In addition to the above-mentioned active ingredients, the pharmaceutical composition according to the present disclosure may contain pharma- ceutically acceptable excipients, additives, buffers, salts for isotonicity adjustment, antioxidants, preservatives, drug stabilizers, and the like. Examples of excipients include, but are not limited to, water, purified water, alcohol, glycerin, lactose, starch, dextrin, white sugar, precipitated silica, honey, rice starch, and tragacanth. In addition, the pharmaceutical composition according to the present disclosure may contain other active ingredients. The amount of each ingredient may be appropriately determined within a medicament-acceptable range. In addition, the dosage of the composition may be appropriately determined depending on the type of drug used and the subject to which it is administered. For example, the active ingredient can be 0.01 to 15% by weight, for example, 0.1 to 5% by weight.

[0040] The route of administration can also be appropriately determined depending on the type of drug used and the subject to which it is administered. The method of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, but suitable examples include intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, intradermal administration, intramuscular administration, eye drops, etc., and among these, intravascular administration is more suitable. The pharmaceutical composition according to the present disclosure may be contained in a wound covering material such as a dressing material. The dressing material is a medical material that can maintain a moist environment and provide an optimal environment for wound healing.

[0041] Examples of mammals include humans, as well as non-human mammals such as monkeys, mice, rats, rabbits, dogs, cats, sheep, goats, alpacas, horses, cattle, pigs, minks, foxes, martens, raccoon dogs, chinchillas, sea otters, otters, beavers, and seals. The dosage can be appropriately determined depending on the type of drug used and the subject to which it is administered. The route of administration can also be appropriately determined depending on the type of drug used and the subject to which it is administered. Preferred routes of administration include, for example, application or spraying of a liquid, lotion, or cream to the skin, or subcutaneous injection, intravenous injection, intrathecal injection, eye drops, and oral administration of a solid or liquid agent. A patch containing the composition according to the present disclosure may be prepared and applied to the skin.

[0042] Also, one embodiment of the present disclosure relates to a method for treating and / or preventing a disease or disorder, comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure to a subject in need of treatment, wherein the disease or disorder may be selected from the group consisting of cancer, a demyelinating disease, an eye disease, and an eye disorder. Still further, another embodiment of the present disclosure relates to the use of a cell culture supernatant according to the present disclosure in the manufacture of a medicament for use in treating and / or preventing a disease or disorder, wherein the disease or disorder may be selected from the group consisting of cancer, a demyelinating disease, an eye disease, and an eye disorder.

[0043] (Cosmetic composition) One embodiment of the present disclosure relates to a cosmetic composition comprising the above-mentioned cell culture supernatant or the above-mentioned cell culture supernatant powder. As shown in Table 3, the cell culture supernatant obtained by the co-culture according to the present disclosure is believed to contain many ingredients that are beneficial to the skin.

[0044] In one embodiment, the cosmetic composition according to the present disclosure is a composition for use in skin care. In some embodiments, the cosmetic composition according to the present disclosure can be used, for example, to improve wrinkles, spots, and / or firmness of the skin, or to promote skin regeneration. In one embodiment, the cosmetic composition according to the present disclosure relates to a composition for application to the skin surface. The composition can be in various product forms, including, but not limited to, solutions, suspensions, lotions, creams, gels, toners, sticks, pencils, sprays, aerosols, ointments, cleansing detergent solutions and cleansing bars, shampoos and hair conditioners, pastes, foams, powders, mousses, shaving creams, wipes, strips, patches, power patches, wound dressings and adhesive bandages, hydrogels, film-forming products, face and skin masks (with and without insoluble sheets), makeup products such as foundations, eyeliners and eyeshadows.

[0045] The cosmetic composition according to the present disclosure may further comprise at least one of a skin anti-aging agent, a skin tone agent, an anti-inflammatory agent, and a sunscreen agent.Skin anti-aging agents include, but are not limited to, peptides having anti-aging activity (WO2014157485A1).Suitable skin tone agents include, but are not limited to, sugar amines, arbutin, deoxyarbutin, hexylresorcinol, kojic acid, hexamidine compounds, salicylic acid, and retinoids including retinol propionate and retinyl propionate.

[0046] Suitable anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory agents (NSAIDS such as ibuprofen, naproxen, etc.), glycyrrhizic acid (also known as glycyrrhetinic acid glycoside) and salts such as dipotassium glycyrrhizinate, glycyrrhetenic acid, licorice extract, bisabolol (e.g., alpha bisabolol), manjistha (extracted from plants of the genus Rubia, particularly Rubia cordifolia), and guggal (extracted from plants of the genus Commiphora, particularly Commiphora mukul), kola extract, chamomile, red clover extract, and whip coral extract (extract from plants of the order Gorgonian), derivatives of any of the above, and mixtures thereof.

[0047] Suitable sunscreens include 2-ethylhexyl-p-methoxycinnamate, 4,4'-t-butylmethoxydibenzoylmethane, 2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid, digalloyl trioleate, 2,2-dihydroxy-4-methoxybenzophenone, ethyl-4-(bis(hydroxypropyl)aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl salicylate, glyceryl-p-amino Benzoate, 3,3,5-tri-methylcyclohexyl salicylate, methyl anthranilate, p-dimethyl-aminobenzoic acid or aminobenzoate, 2-ethylhexyl-p-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonic acid, 2-(p-dimethylaminophenyl)-5-sulfonebenzoxazoic acid, octocrylene, zinc oxide, benzylidene camphor and its derivatives, titanium dioxide, and mixtures thereof.

[0048] The cosmetic composition according to the present disclosure may further comprise at least one additive that is conventionally used in the cosmetic field and does not affect the properties of the composition according to the present disclosure, such as a thickener, a fragrance, a pearlescent agent, a preservative, a sunscreen, an anionic or nonionic or cationic or amphoteric polymer, a protein, a protein hydrolysate, such as 18-methyl eicosanoic acid, a vitamin, panthenol, a silicone, a fatty acid such as a vegetable oil, an animal oil, a mineral oil or a synthetic oil, a gelling agent, an antioxidant, a solvent, a filter, a scrub, etc. The cosmetic ingredients may also include cosmetic ingredients such as cleaning agents, odor absorbers, colorants, abrasives, absorbents, fragrances, dyes, pigments / colorants, essential oils, anti-caking agents, defoamers, antimicrobial agents, binders, biological additives, buffers, fillers, chelating agents, chemical additives, cosmetic astringents, cosmetic biocides, denaturants, drug astringents, emollients, topical analgesics, film formers or materials, opacifiers, pH adjusters, preservatives, propellants, reducing agents, scavengers, skin cooling agents, skin protective agents, thickeners, viscosity regulators, vitamins, and combinations thereof. These additives may be present in the composition according to the present disclosure in a proportion that is not limited, but is preferably or advantageously in the range of 0 to 50% by weight, 5 to 40% by weight, or 30 to 50% by weight, based on the total weight of the composition.

[0049] The cosmetic compositions according to the present disclosure, for topical application to the skin, may in particular have the form of an aqueous or oily solution, a dispersion of lotion or serum type, an emulsion with a liquid or semi-liquid consistency of milk type obtained by dispersion of a fatty phase in an aqueous phase (O / W) or vice versa (W / O), a suspension or emulsion with a soft consistency of aqueous or anhydrous gel or cream type, or else in the form of microcapsules or microparticles, vesicular dispersions of ionic and / or nonionic type, or foams. These compositions are prepared according to the usual methods. The compositions preferably have an aqueous phase between 5 and 99.5%.

[0050] The pH of the cosmetic composition according to the present disclosure is not limited, but is generally between 2 and 12, preferably between 3 and 9. The pH can be adjusted to the target value by adding a base (organic or inorganic) to the composition, for example, ammonia, sodium hydroxide, potassium hydroxide, or a primary, secondary or tertiary (poly)amine, such as monoethanolamine, diethanolamine, triethanolamine, isopropanolamine or 1,3-propanediamine, or with a basic amino acid or polyamino acid, such as lysine or arginine, or by adding an inorganic or organic acid, preferably a carboxylic acid, such as, for example, citric acid.

[0051] The cosmetic compositions according to the present disclosure in particular constitute creams (e.g. day creams, night creams, anti-aging creams, moisturizing creams, make-up removal creams, foundation creams or sun creams) for washing, protecting, treating or caring for the face, hands, feet, major anatomical folds or the body, liquid foundations, make-up removal milks, protective or caring body milks, sun milks, lotions, gels or foams for caring for the skin, such as cleansing lotions, sun lotions, artificial tanning lotions, bath compositions, deodorant compositions containing a disinfectant, aftershave gels or lotions, depilatory creams, compositions for combating insect bites.

[0052] The cosmetic composition according to the present disclosure may be applied at least once a day, twice a day, or more frequently during the treatment period. When applied twice a day, the first and second applications are separated, for example, by at least 1 to 12 hours. Typically, the composition may be applied in the morning and / or at night before going to bed.

[0053] In addition, one embodiment of the present disclosure relates to a cosmetic method comprising administering or applying an effective amount of the cosmetic composition according to the present disclosure to a subject in need of treatment. Further, one embodiment of the present disclosure relates to the use of the cell culture supernatant according to the present disclosure in the manufacture of a cosmetic product.

[0054] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0055] <Example 1: Co-culture of MSCs> Human adipose-derived MSC cells, human umbilical cord-derived MSC cells, and human amnion-derived MSC cells were cultured in the following manner. 2 The cells were seeded in the medium and cultured in MSC culture medium. The medium was changed every 2-3 days. Adipose-derived MSCs, umbilical cord-derived MSCs, and amnion-derived MSCs were passaged multiple times, after which the cells were detached by enzyme treatment and seeded under the following 6 conditions.

[0056] 1) Human adipose-derived MSC cells at 5,000 cells / cm 2 Group seeded at a cell density of 2) Human umbilical cord-derived MSC cells at 5,000 cells / cm 2 Group seeded at a cell density of 3) Human amnion-derived MSC cells at 5,000 cells / cm 2 Group seeded at a cell density of 4) Adipose MSCs and umbilical cord MSCs were cultured at a ratio of 1:1, with a total of 5,000 cells / cm 2 Group seeded at a cell density of 5) Umbilical cord MSCs and amniotic membrane MSCs were cultured at a ratio of 1:1, with a total of 5,000 cells / cm 2 Group seeded at a cell density of 6) Adipose MSCs and amniotic MSCs were cultured at a ratio of 1:1, with a total of 5,000 cells / cm 2 Group seeded at a cell density of

[0057] These cells were seeded, and when they reached confluence, the medium was removed, and the cells were washed twice with PBS, after which basal medium was added. After culturing with the addition of basal medium, the medium was collected and the cell culture supernatant was obtained. The supernatant was sterilized by filtration using a 0.22 μm filter, and then stored at -80°C.

[0058] <Example 2: Proteome analysis of supernatant> Promega Corporation (Tokyo) was commissioned to perform proteome analysis of the components in the collected supernatant. As a result, as shown in Table 1, several components were identified that were not present in the cell culture supernatant when adipose-derived MSCs, umbilical cord-derived MSCs, and amniotic membrane-derived MSCs were cultured individually, but were present only when co-culture was performed. Table 1 summarizes the quantitative values ​​of each protein present in the cell culture supernatant when adipose-derived MSCs, umbilical cord-derived MSCs, and amniotic membrane-derived MSCs were cultured individually, and when co-cultured in combinations of adipose x umbilical cord, umbilical cord x amniotic membrane, and adipose x amniotic membrane. This result indicates that the cell culture supernatant obtained by co-culture has different activities from the cell culture supernatant obtained by conventional single culture, and is useful as a pharmaceutical or cosmetic. Tables 2 and 3 summarize the pharmacological and cosmetic effects of some of these components (GCLC, RAD21, RND3).

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] Although preferred embodiments of the present invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only, and that various modifications, changes, and substitutions may be made by those skilled in the art without departing from the present invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the present invention. In addition, the contents of all publications, including patents and patent applications, referenced in this specification should be construed as being incorporated by reference as if set forth herein. [Industrial Applicability]

[0063] The present inventors have found that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant with unexpectedly excellent activity can be obtained. For example, by co-culturing two of MSCs derived from fat, umbilical cord, or amniotic membrane, a supernatant containing components not contained in the culture supernatant of each MSC alone can be obtained. The components obtained by co-culturing include those that have useful efficacy as pharmaceuticals or cosmetics, and can be used industrially.

Claims

1. A method for producing a mesenchymal stem cell culture supernatant, the method being for producing a pharmaceutical composition for use in treating, preventing, or ameliorating a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases, or for producing a cosmetic composition for use in tissue regeneration or repair, wound healing, or angiogenesis, comprising: i) Co-culturing two or more types of mesenchymal stem cells; and ii) Recovering the culture supernatant Including, However, with regard to the method for producing a mesenchymal stem cell culture supernatant for producing a cosmetic composition for use in tissue regeneration or repair, wound healing, or angiogenesis, the following applies, except that the two or more types of mesenchymal stem cells are a combination of only dental pulp-derived cells and hair follicle-derived cells: A method for producing a mesenchymal stem cell culture supernatant for producing a cosmetic composition for use in tissue regeneration or repair, wound healing, or angiogenesis, excluding a method in which the two or more types of mesenchymal stem cells are a combination of only cells derived from the umbilical cord or umbilical cord blood and cells derived from the placenta.

2. A method for producing a mesenchymal stem cell culture supernatant, comprising: i) Co-culturing two or more types of mesenchymal stem cells; and ii) Recovering the culture supernatant Including, the mesenchymal stem cells are cells derived from a tissue selected from the group consisting of adipose tissue, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, and hair follicles; However, unless the two or more types of mesenchymal stem cells are a combination of only dental pulp-derived cells and hair follicle-derived cells, Unless the two or more types of mesenchymal stem cells are a combination of only cells derived from the umbilical cord or umbilical cord blood and cells derived from the placenta, A method other than the case where the two or more types of mesenchymal stem cells are a combination of only adipose-derived cells and umbilical cord-derived cells.

3. The method according to claim 1 or 2, further comprising culturing the two or more types of mesenchymal stem cells individually prior to co-culturing.

4. The method according to claim 1 or 2, wherein two types of mesenchymal stem cells are used.

5. The method according to claim 1 or 2, wherein human adipose stem cells and human amniotic stem cells, or human umbilical cord stem cells and human amniotic stem cells are used.

6. 3. The method according to claim 1, wherein the mesenchymal stem cell culture supernatant contains a component that is not present when the two or more types of mesenchymal stem cells are cultured individually.

7. The method of claim 1 or 2, wherein the mesenchymal stem cell culture supernatant contains a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), glutamate-cysteine ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3).

8. The method of claim 1 or 2, wherein the mesenchymal stem cell culture supernatant contains an increased amount of a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), glutamate-cysteine ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3) compared to the amount contained when the two or more types of mesenchymal stem cells are cultured individually.

9. 3. The method of claim 1 or 2, comprising culturing the cells in a serum-free medium that maintains the undifferentiated state of MSCs, a medium in which serum is added to a basal medium, a culture medium for human mesenchymal stem cells, a feederless medium for mesenchymal stem cells, and / or a chemically defined serum-free medium.

10. A cell culture supernatant obtained by the method according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising a cell culture supernatant obtained by the method according to any one of claims 1 to 9.

12. A cosmetic composition comprising a cell culture supernatant obtained by the method according to any one of claims 1 to 9.

13. A method for producing a mesenchymal stem cell culture supernatant powder, comprising freeze-drying the cell culture supernatant obtained by the method according to any one of claims 1 to 9.

14. A mesenchymal stem cell culture supernatant powder obtained by the method of claim 13.

15. A pharmaceutical composition comprising a mesenchymal stem cell culture supernatant powder obtained by the method of claim 13.

16. A cosmetic composition comprising a mesenchymal stem cell culture supernatant powder obtained by the method of claim 13.

17. 12. The pharmaceutical composition of claim 11 for use in the treatment, prevention, or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases.

18. 16. The pharmaceutical composition of claim 15 for use in the treatment, prevention, or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases.

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