Composition comprising mesenchymal stem cell-conditioned medium derived from wharton's jelly and use thereof for wound healing or skin improvement

Abstract

The present invention relates to a composition for wound healing or skin improvement, comprising a mesenchymal stem cell-conditioned medium derived from Wharton's jelly. The composition of the present invention is a stem cell-conditioned medium that does not contain components derived from mammals other than humans, and ensures stability when used as cosmetics or medicines. In addition, the composition promotes the secretion of stem cell secretome by regulating the rigidity of a substrate, and thus is effective in wound healing or skin improvement.

Description

Medium composition conditioned with mesenchymal stem cells derived from Wharton's jelly and composition thereof for wound healing or skin improvement

[0001] This application claims priority to Korean Patent Application No. 10-2023-0177130, filed December 7, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a medium conditioned with mesenchymal stem cells derived from Wharton's jelly, a method for preparing the same, and a use thereof for wound healing or skin improvement.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells with multipotency and self-renewal capabilities. Researchers have reported that MSCs secrete various cytokines, growth factors, and chemokines in response to environmental signals, thereby exhibiting wound healing effects (e.g., regulating the microenvironment of damaged tissues by suppressing inflammation and immunomodulating effects) or skin improvement effects (e.g., improving skin elasticity and preventing wrinkles). Skin can develop complications such as bacterial infections due to external force-induced wounds (meaning damage or loss of the skin or surrounding tissues due to physical injury), which, if aggravated, can lead to acute or chronic wounds. Furthermore, skin can experience a decrease in elasticity and the formation of wrinkles due to "intrinsic aging" caused by aging and "extrinsic aging" caused by external factors such as UV rays and pollution.

[0004] Mesenchymal stem cells have shown significant potential in regenerative medicine and the cosmetics industry. However, long-term culture (proliferation) has been reported to impede the secretion of cytokines, growth factors, and other secretory activities, resulting in reduced tissue regeneration potential. Furthermore, research on the role of the mechanical or chemical microenvironment, particularly stiffness, in mesenchymal stem cell culture remains limited.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] (Patent Document 1) Korean Patent No. 10-2456576

[0008] [Non-patent literature]

[0009] (Non-patent literature 1) Park Se-ah, et al. “Analysis of changes in characteristics of umbilical cord-derived stem cells following subculture.” Korean Journal of Reproductive Medicine: Vol. 36.1 (2009).

[0010] (Non-patent Document 2) Guimaraes, Carlos F., et al. “The stiffness of living tissues and its implications for tissue engineering.” Nature Reviews Materials 5.5 (2020): 351-370.

[0011] The present invention aims to solve one or more of the problems of the above-mentioned prior art.

[0012] The present invention aims to provide a medium composition conditioned with stem cells that exhibits excellent skin improvement effects, a method for producing the same, and a cosmetic composition, pharmaceutical composition, or application product using the same.

[0013] The purpose of the present invention is not limited to the purposes mentioned above. The purpose of the present invention will become more apparent from the following description, and may be realized by the means and combinations thereof set forth in the claims.

[0014] A representative configuration of the present invention to achieve the above purpose is as follows.

[0015] In one aspect of the present invention, a medium composition conditioned with mesenchymal stem cells obtained by a method including culturing mesenchymal stem cells on a low-rigidity substrate is provided, which medium composition exhibits wound healing, regeneration, wrinkle improvement, anti-aging or reverse-aging effects on the skin.

[0016] In some embodiments, the culture of mesenchymal stem cells may be performed in a culture medium comprising one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin.

[0017] In some embodiments, the low stiffness substrate may be a substrate having a stiffness of 10 kPa to 3.5 GPa.

[0018] In some embodiments, the low stiffness substrate may be a substrate having a stiffness of 10 kPa to 1 GPa.

[0019] In some implementations, the low stiffness substrate may be a substrate having a stiffness of 50 kPa to 1000 kPa.

[0020] In some embodiments, the medium composition may exhibit one or more effects selected from promoting skin cell proliferation, promoting elastin synthesis, promoting fibronectin synthesis, promoting collagen III synthesis, and inhibiting aging gene expression.

[0021] In some embodiments, the skin cells may be fibroblasts.

[0022] In some embodiments, the mesenchymal stem cells may be derived from human umbilical cord Wharton's jelly.

[0023] In another aspect of the present invention, a cosmetic composition comprising an effective amount of the badge composition of the present invention is provided.

[0024] In some embodiments, the cosmetic composition may have a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays.

[0025] In another aspect of the present invention, a pharmaceutical composition for treating or preventing skin damage is provided, comprising an effective amount of the badge composition of the present invention.

[0026] In another aspect of the present invention, a pharmaceutical product for preventing or treating skin damage is provided, comprising an effective amount of the badge composition of the present invention.

[0027] In another aspect of the present invention, a skin external preparation for preventing or treating skin damage is provided, which comprises an effective amount of the badge composition of the present invention.

[0028] In another aspect of the present invention, a method for preparing a medium composition conditioned with mesenchymal stem cells is provided, comprising the steps of culturing mesenchymal stem cells on a low-rigidity substrate and recovering a culture medium from the culture.

[0029] In some embodiments, the low stiffness substrate may be a substrate having a substrate stiffness of 10 kPa to 3.5 GPa.

[0030] In some embodiments, the low stiffness substrate may be a substrate having a substrate stiffness of 10 kPa to 1 GPa.

[0031] In some embodiments, the low stiffness substrate may be a substrate having a substrate stiffness of 50 kPa to 1000 kPa.

[0032] In some embodiments, the mesenchymal stem cells may be isolated from human umbilical Wharton jelly.

[0033] In some embodiments, the culturing of mesenchymal stem cells may be performed in a culture medium comprising one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin.

[0034] The present invention is based, at least in part, on the discovery that a medium composition (a medium composition conditioned with mesenchymal stem cells) obtained by culturing mesenchymal stem cells (e.g., mesenchymal stem cells derived from Wharton's jelly) under controlled micro-culture conditions (e.g., substrate stiffness and / or culture medium) exhibits excellent skin improvement effects (e.g., wound healing, skin regeneration, wrinkle improvement, skin anti-aging, skin reverse-aging, etc.). The medium composition of the present invention does not contain any components derived from mammals other than human, thereby ensuring safety when used as a cosmetic or pharmaceutical composition applied to the skin. In addition, the medium composition of the present invention exhibits wound healing (e.g., promotion of fibroblast proliferation) or skin improvement (e.g., promotion of skin cell growth, elastin synthesis, fibronectin synthesis, or collagen III synthesis, and inhibition of aging gene expression) effects by containing the secretome secreted when culturing mesenchymal stem cells under controlled micro-culture conditions.

[0035] Figures 1a and 1b illustrate the results of confirming the fibroblast proliferation effect of a medium composition conditioned with mesenchymal stem cells according to one embodiment of the present invention. Figure 1a is a microscopic photograph confirming the growth of fibroblasts, and Figure 1b is a graph quantitatively representing the number of cells in a region of interest (ROI) spot.

[0036] Figures 2a to 2c show the results of confirming the wound healing effect of a medium composition conditioned with mesenchymal stem cells according to one embodiment of the present invention. Figure 2a shows a schematic diagram of attaching or removing a polydimethylsiloxane (PDMS) rubber block (20 mm × 6 mm × 4 mm) to or from the bottom of a 6-well plate. Figure 2b is a microscopic photograph confirming the proliferation of fibroblasts for 11 days after removing the rubber block, and Figure 2c is a graph quantitatively showing the distance (μm) between linear cell walls.

[0037] Figures 3a to 3e show the results of confirming the skin improvement effect of a medium composition conditioned with mesenchymal stem cells according to an embodiment of the present invention. Figure 3a is a graph comparing the growth of skin cells in a 10% FBS medium (positive control), a medium conditioned with mesenchymal stem cells according to an embodiment of the present invention, and DMEM-CM (negative control). Figures 3b to 3d show the results of confirming the expression levels of elastin genes, fibronectin genes, and collagen III genes, respectively, to confirm the promotion of extracellular matrix (ECM) production. Figure 3e is a graph confirming the expression level of the p16 gene, which is an aging gene.

[0038] The detailed description of the present invention described below will be described with reference to specific embodiments in which the present invention may be practiced (if any), with reference to the drawings, but the present invention is not limited thereto, but is defined only by the appended claims to the full scope equivalent to or equivalent to what the claims describe. It should be understood that the various embodiments / embodiments of the present invention, while different from each other, are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment / embodiment to another, or multiple embodiments / embodiments may be combined, without departing from the spirit and scope of the present invention. Technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly used in the art to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and terms expressed in the singular should be construed to also refer to the plural (i.e., at least one), unless the context makes it inappropriate.

[0039] The term "about" as used herein refers to the typical error range for each value known to those of ordinary skill in the art. Furthermore, unless otherwise specified, all numbers, values, and / or expressions expressing ingredients, conditions, compositions, amounts, and so forth used herein should be understood as being modified by the term "about" because such numbers are approximations that inherently reflect various uncertainties in the measurement of obtaining such values, among other things.

[0040] The present invention is based, at least in part, on the discovery that a medium composition obtained by culturing mesenchymal stem cells (e.g., mesenchymal stem cells derived from Wharton's jelly) under controlled micro-culture conditions (e.g., substrate stiffness and / or culture medium) exhibits excellent skin improvement effects (e.g., wound healing, skin regeneration, wrinkle improvement, skin anti-aging, skin reverse aging, etc.). The medium composition of the present invention does not contain any components derived from mammals other than human, thereby ensuring safety when used as a cosmetic or pharmaceutical composition applied to the skin. In addition, the medium composition of the present invention exhibits wound healing (e.g., promoting fibroblast proliferation) or skin improvement (e.g., promoting skin cell growth, elastin synthesis, fibronectin synthesis, or collagen III synthesis, and inhibiting the expression of aging genes) effects by containing a secretome secreted when culturing mesenchymal stem cells under controlled micro-culture conditions (particularly, low-stiffness substrate and / or medium).

[0041] In one aspect of the present invention, a medium composition conditioned with mesenchymal stem cells obtained by a method comprising culturing mesenchymal stem cells on a low-rigidity substrate is provided.

[0042] As used herein, the term "medium conditioned with mesenchymal stem cells" refers to a stem cell culture medium or a supernatant thereof containing various secretomes (e.g., cell-derived proteins such as cytokines and growth factors) secreted from the stem cells during the culturing of the mesenchymal stem cells. The stem cell-conditioned medium can be obtained by culturing stem cells in a culture medium for a predetermined period of time. Generally, the stem cell-conditioned medium refers only to the culture medium remaining after removing cells or tissues, and may contain secreted cytokines, chemokines, growth factors, etc. The obtained conditioned medium can be used as is or after one or more separation, concentration, and / or purification steps.

[0043] The term "mesenchymal stem cell" or "MSC" refers to a stem cell with multipotency and self-renewal capacity that can differentiate into various cell types (e.g., adipocytes, chondrocytes, skin cells, osteocytes, etc.). MSC is used interchangeably with "mesenchymal stromal cells."

[0044] Mesenchymal stem cells can be either "autologous" or "allogeneic." The term "autologous" means that the source of the cells is the patient's own cells, such as cells isolated from the patient's own bone marrow or adipose tissue. In contrast, "allogeneic" means that the source of the cells is cells obtained from a donor. Allogeneic MSCs can be derived from, but are not limited to, the donor's bone marrow, adipose tissue, umbilical cord tissue, or blood. Alternatively, allogeneic MSCs can be derived from the donor via induced pluripotent stem cells (iPSCs). As another alternative, allogeneic MSCs can be derived from embryonic stem cells (ESCs). Preferably, the mesenchymal stem cells of the present invention are allogeneic MSCs derived from human umbilical cord Wharton's jelly.

[0045] In some embodiments, the medium (for conditioning) of the mesenchymal stem cells of the present invention may include various basal defined media suitable for stem cell culture. Basal defined media suitable for stem cell culture are well known in the art and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), F12, or RPMI. Basal defined media are chemically defined basal media that may include glucose, essential amino acids (e.g., glutamine, lysine, methionine, alanine, arginine, etc.), vitamins (e.g., vitamins A, B, C, E, riboflavin, thiamine, etc.), inorganic compounds (e.g., calcium, magnesium, potassium, sodium, chloride, phosphate, bicarbonate, etc.), acids (e.g., pyruvate), proteins (e.g., insulin, etc.). In some embodiments, the culture medium for conditioning with mesenchymal stem cells may further comprise one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin. Preferably, the culture medium for conditioning with mesenchymal stem cells may contain transferrin, selenium, insulin, glutathione, phospholipids, and human albumin. More preferably, the culture medium for conditioning with mesenchymal stem cells may be a basal defined medium supplemented with transferrin, selenium, insulin, glutathione, phospholipids, and human albumin.

[0046] In some embodiments, the culture medium for conditioning with mesenchymal stem cells may further comprise one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin in addition to the components typically included in an animal cell culture medium. The components typically included in an animal cell culture medium may include glucose, L-glutamine, sodium pyruvate, calcium, magnesium, potassium, sodium, chloride, phosphate, bicarbonate, amino acids (e.g., L-lysine, L-methionine, etc.), vitamins (e.g., riboflavin, thiamine, etc.), but the components or contents may be appropriately changed depending on the type of cells to be cultured or the purpose of the experiment. In some embodiments, a conditioned medium derived from a medium comprising one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin may exhibit superior wound healing (e.g., promoting fibroblast proliferation) or skin improvement (e.g., promoting skin cell growth, elastin synthesis, fibronectin synthesis, or collagen III synthesis, and inhibiting expression of aging genes) effects than a conventional stem cell culture medium.

[0047] In some embodiments, the mesenchymal stem cells of the present invention can be cultured in a chemically defined medium. The culture medium obtained by culturing mesenchymal stem cells in a chemically defined medium is referred to as a chemically defined conditioned medium (CDCM). The term "chemically defined medium" refers to a growth medium suitable for in vitro cell culture of human or animal cells, in which all chemical components are known. Standard cell culture media typically consist of media supplemented with animal serum (e.g., FBS) as a source of nutrients and other less well-defined elements, whereas chemically defined media contain only recombinant proteins and / or hormones. Therefore, chemically defined media or chemically cultured conditioned media not only aid in studying the interactions between molecules and cells, but also eliminate risk factors such as animal-derived viruses and mycoplasmas that may be transmitted to the final product during cosmetic and / or pharmaceutical development. In some embodiments, the medium composition of the present invention is a chemically defined conditioned medium.

[0048] In some embodiments, the low stiffness substrate can have a stiffness of from 10 kPa to 3.5 GPa, specifically from 10 kPa to 1 GPa, more specifically from 50 kPa to 1000 kPa, and even more specifically from 500 kPa.

[0049] In some embodiments, the mesenchymal stem cells may be derived from human umbilical cord Wharton's jelly. The term "umbilical cord" is used interchangeably with "umbilical cord" and may refer to the cord that connects the mother's abdomen to the fetus's abdomen, allowing the mammalian fetus to develop in the placenta, and generally refers to a tissue composed of three blood vessels, namely two umbilical arteries and one umbilical vein, surrounded by Wharton's jelly.

[0050] In some embodiments, the mesenchymal stem cells may be mesenchymal stem cells obtained by passage culture. For example, the mesenchymal stem cells may be obtained by passage culture 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 or more times. The passage culture may be performed for a number of times between two ranges selected from the above values. Preferably, the mesenchymal stem cells may be obtained by passage culture 3 to 10 times or 3 to 7 times.

[0051] In some embodiments, the medium composition conditioned by the mesenchymal stem cells of the present invention can be obtained by culturing mesenchymal stem cells until confluent (primary culture) and further culturing the mesenchymal stem cells thus cultured in a chemically defined medium (for conditioning) (secondary culture). In some embodiments, the primary culture can be performed using a complete culture medium for animal cell culture known in the art to facilitate the proliferation of mesenchymal stem cells. In some embodiments, the primary culture can include subculture. In some embodiments, the secondary culture can be performed for 1 to 20 days, 1 to 15 days, 3 to 15 days, or 3 to 10 days.

[0052] In some embodiments, the composition of the present invention exhibits wound healing, regeneration, wrinkle improvement, anti-aging or reverse-aging effects on the skin.

[0053] In some embodiments, the medium composition of the present invention exhibits one or more of the effects of promoting skin cell proliferation, promoting elastin synthesis, promoting fibronectin synthesis, promoting collagen III synthesis, and inhibiting aging gene expression.

[0054] In some embodiments, the skin cells can be fibroblasts from a human or non-human mammal.

[0055] In some embodiments, the medium conditioned with the mesenchymal stem cells of the present invention or cells differentiated therefrom may be used for wound healing or improving skin conditions or skin damage. Wound healing or improving skin conditions or skin damage may include one or more of the following: formation of skin tissue, wound relief, regeneration or elasticity enhancement, anti-aging, wrinkle improvement, moisturizing, and barrier strengthening.

[0056] In some embodiments, the medium conditioned with the mesenchymal stem cells of the present invention or cells differentiated therefrom may be used for the purpose of healing skin wounds. The term "wound" refers to an injury to the human body resulting from a cell or tissue being cut, torn, broken, burned, or traumatized, or from a disorder or disease that causes such injury. In some embodiments, the wound may include damage to one or more of the epidermis, dermis, or subcutaneous fat layers of the skin. Additionally, the wound may be caused by a cut, incision, puncture wound, abrasion, contusion, puncture wound, laceration, tear, fracture, burn, amputation, or a combination thereof.

[0057] In some embodiments, the wound or skin damage may be an inflammatory injury, including inflammation due to dermatitis, atopic dermatitis, eczema, bacterial infection, viral infection, or fungal infection, burns, inflammation due to burns, wounds, or inflammation due to wounds. Furthermore, the skin damage may be skin pigmentation, including freckles, lentigines, lentigos, nevi, melanoma, drug-induced pigmentation, post-inflammatory pigmentation, and pigmentation resulting from dermatitis.

[0058] In some embodiments, the medium conditioned with the mesenchymal stem cells of the present invention or cells differentiated therefrom may be used for medical skin regeneration (e.g., treating, alleviating, or improving dermatitis, infectious inflammation, burns, pigmentation, wounds, etc.) or cosmetic skin regeneration.

[0059] In some embodiments, a medium conditioned with the mesenchymal stem cells of the present invention or cells differentiated therefrom may be used for improving skin wrinkles.

[0060] In some embodiments, media conditioned with the mesenchymal stem cells of the present invention or cells differentiated therefrom may be used for anti-aging or reverse-aging purposes.

[0061] In some embodiments of the present invention, the medium conditioned with human umbilical cord Wharton's jelly-derived mesenchymal stem cells, specifically the medium conditioned with human umbilical cord Wharton's jelly-derived mesenchymal stem cells cultured on a substrate having a stiffness of 500 kPa, significantly enhanced the proliferation of human neonatal dermal fibroblasts, exhibiting significantly higher cell proliferation compared to the control group (see Example 2.2), and exhibited excellent in vitro wound healing effects (see Example 2.3).

[0062] In addition, in some embodiments of the present invention, a medium conditioned with human umbilical cord Wharton's jelly-derived mesenchymal stem cells, specifically a medium conditioned with human umbilical cord Wharton's jelly-derived mesenchymal stem cells cultured on a 500 kPa substrate, exhibited a skin improvement effect of promoting the growth of young skin cells, increasing the expression levels of extracellular matrix elastin, fibronectin, and collagen III, and suppressing the expression of an aging gene (p16) (see Example 3).

[0063] In another aspect of the present invention, a cosmetic composition is provided comprising an effective amount of a medium composition as disclosed herein.

[0064] In some embodiments, the cosmetic composition may have a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays.

[0065] The cosmetic composition of the present invention may contain, in addition to the medium composition conditioned with stem cells as an active ingredient, components commonly used in cosmetic compositions, and such components include, for example, conventional auxiliary agents such as (additional) antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and / or carriers.

[0066] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured into the formulation of a flexible toner (skin), a nourishing toner (milk lotion), a nourishing cream, a massage cream, an essential oil, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0067] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0068] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0069] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0070] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. may be used as a carrier component.

[0071] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0072] The carrier component may be included in an amount of about 1 to about 99.99 wt%, preferably about 80 to about 90 wt%, based on the total weight of the cosmetic composition.

[0073] In another aspect of the present invention, a pharmaceutical composition is provided comprising an effective amount of a medium composition as disclosed herein. The term "veterinary composition" may be used interchangeably with the term "medical composition" when applied to animals other than humans.

[0074] The pharmaceutical composition of the present invention may further comprise one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents. The pharmaceutically acceptable carriers, excipients and / or diluents may be those commonly used in the art. Examples of the carriers, excipients or diluents include, but are not limited to, mineral oils such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and silicon dioxide.

[0075] When formulating, additives such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants can be used. The additives for the formulation can be selected from those commonly used in the pharmaceutical field.

[0076] In addition, the pharmaceutical composition of the present invention may be formulated in a desirable form depending on the method of use, and may be formulated by adopting a method known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Specific examples of such formulations include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonades, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0077] Furthermore, the pharmaceutical composition of the present invention may be preferably formulated using any suitable method known in the art or using the method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0078] In some embodiments, the pharmaceutical composition may be for the prevention or treatment of skin damage as disclosed herein.

[0079] In another aspect of the present invention, a quasi-drug comprising an effective amount of a medium composition as disclosed herein is provided. The term "quasi-drug" as used herein refers to products that have a milder effect than a pharmaceutical product among products used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing a disease in humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for the purpose of pharmaceutical products, and include fiber and rubber products used for the treatment or prevention of diseases in humans or animals, products that have a mild or no direct effect on the human body, are not instruments or machines, and similar products, and sterilizers and insecticides for preventing infectious diseases.

[0080] The type or formulation of the pharmaceutical composition of the present invention is not particularly limited, but may be a bandage, gauze, cotton, adhesive bandage, disinfectant, shower foam, gargle, wet tissue, detergent soap, hand wash, humidifier filler, mask, mask pack, or filter filler.

[0081] When the composition of the present invention is incorporated into an over-the-counter drug for the purpose of wound healing or improving skin damage or condition, the composition may be used as is or in combination with other over-the-counter drug ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined depending on the intended use.

[0082] In another aspect of the present invention, a topical skin preparation comprising an effective amount of a medium composition as disclosed herein is provided. The topical skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a lotion, or a combination thereof. The topical skin preparation may be appropriately blended with ingredients commonly used in topical skin preparations such as cosmetics or pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof, as needed.

[0083] In some embodiments, the skin includes all skin areas of the body, including the face, hands, arms, legs, feet, chest, stomach, back, buttocks, and scalp.

[0084] In another aspect of the present invention, a method for preparing a medium composition conditioned with mesenchymal stem cells as disclosed herein is provided. Specifically, the method comprises culturing mesenchymal stem cells on a low-rigidity substrate and recovering a culture medium from the culture.

[0085] In some embodiments, the manufacturing method of the present invention may further comprise the step of obtaining human umbilical cord Wharton's jelly and isolating mesenchymal stem cells therefrom. Human umbilical cord Wharton's jelly may refer to Wharton's jelly derived from the umbilical cord separated from the mother after birth. The separated umbilical cord may be promptly stored in a sterilized container and on ice after separation.

[0086] Methods for isolating stem cells by obtaining human umbilical cord Wharton's jelly are well known in the art and can be appropriately performed by a person skilled in the art depending on the purpose of culture. For example, a method for isolating stem cells by obtaining Wharton's jelly may include a step of removing external blood from an isolated umbilical cord and a step of removing an artery and vein of the umbilical cord from which the blood has been removed, and may further include a step of cutting the umbilical cord from which the artery and vein have been removed into pieces of a certain size (e.g., 1 to 20 mm). Removal of blood may be performed through a process of removing blood, contaminants, etc. present in the tissue by washing once, twice, or three or more times with phosphate buffered saline (PBS) containing an antibiotic, such as penicillin, streptomycin, gentamicin, or a combination thereof.

[0087] The step of isolating mesenchymal stem cells may be performed by treating the umbilical cord (e.g., the separated umbilical cord) with a separation enzyme. The step of treating the separation enzyme may include reacting the separation enzyme directly with the umbilical cord, or further cutting the umbilical cord into smaller pieces using sterilized scissors or the like, and then reacting the cut cells with the separation enzyme. The separation enzyme may be, but is not limited to, collagenase, which is an enzyme that breaks down peptide bonds in collagen (e.g., collagenase type I, type II, type III, type IV, or a combination thereof), trypsin, dispase, or a combination thereof. Additionally, the step of isolating mesenchymal stem cells may further include a step of inactivating the separation enzyme, for example, by adding serum to stop the enzymatic reaction.

[0088] In some embodiments, the manufacturing method of the present invention may further comprise a step of passage the isolated mesenchymal stem cells. For example, the isolated mesenchymal stem cells may be passaged 1 to 10 times. The culture medium for the mesenchymal stem cells may be a xeno-free medium containing human platelet lysate. In some embodiments, the mesenchymal stem cells may be passaged 3 to 7 times in the xeno-free medium.

[0089] The term "xeno-free" means free of material of non-human animal origin. For example, when stem cells are cultured in a xeno-free medium, the medium means a culture medium that does not contain material of non-human animal origin (i.e., xenogeneic) (i.e., contains only material of the same species, i.e., human origin).

[0090] Xenogeneic culture media for in vitro culture, induction, maintenance, proliferation and differentiation of mesenchymal stem cells may include, but are not limited to, KnockOut Dulbecco's Modified Eagle's Medium (KO-DMEM), Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, HyQ ADCF-MAb (HyClone), DMEM F12 and combinations thereof. In some embodiments, the xenogeneic culture media of the present invention may be DMEM F12.

[0091] In some embodiments, the manufacturing method of the present invention may comprise culturing passaged mesenchymal stem cells on a low-stiffness substrate as disclosed herein.

[0092] Stem cells can change their behavioral patterns (e.g., cell division, differentiation, etc.), gene expression patterns, and secreted factors depending on their surrounding microenvironment. In particular, the secretome secreted by stem cells, the lineage of cells differentiated, and the rate of cell division can change depending on the rigidity of the substrate on which they are cultured or the culture medium (type and content of components, etc.). The mechanical properties of materials (e.g., the substrate of a cell culture plate), particularly the rigidity, are related to the load and deformation of cells (related to the elastic modulus of cells), and several studies have shown that mechanical properties significantly affect the differentiation process of stem cells. Biological tissues are fundamentally composed of the extracellular matrix (ECM) and cells, which interact with the microenvironment to regulate the secretome of cells and determine cell fate. The researchers of the present invention confirmed that when human umbilical cord Wharton jelly-derived mesenchymal stem cells are cultured on a substrate having a stiffness of about 500 kPa or about 3 GPa, particularly on a substrate having a stiffness of about 500 kPa, they can heal wounds or improve skin damage through one or more of the effects of promoting cell proliferation, promoting elastin synthesis, promoting fibronectin synthesis, promoting collagen III synthesis, and suppressing aging gene expression.

[0093] The stiffness of the substrate can be controlled by coating the bottom of the plate on which cells are cultured with a soft biomaterial such as a hydrogel, a protein, or a nanoparticle, but is not limited thereto. The material constituting the hydrogel is a natural polymer, a synthetic polymer, or a hybrid thereof. The natural polymer may include, but is not limited to, collagen, elastin, fibrin, silk, Matrigel, hyaluronic acid (HA), chitosan, collagen, laminin, gelatin, etc. The synthetic polymer may include, but is not limited to, PHEMA (poly hydroxyethyl methacrylate), PHPMA (poly hydroxypropyl methacrylate), PPF (poly propylene fumarate), PVA (poly vinyl alcohol), PEG (poly ethylene glycol), PEGMA (poly ethylene glycol monoacrylate), PAAM (polyacrylamide), etc. In addition, the hybrid may be a component obtained by mixing or copolymerizing a natural polymer and a synthetic polymer.

[0094] In some embodiments, the manufacturing method of the present invention may further comprise a step of filtering, concentrating, or purifying the culture medium of mesenchymal stem cells after recovery. The stem cell culture medium may be obtained during or after culturing mesenchymal stem cells, and may be obtained as is or as a concentrate obtained by concentrating the culture medium or supernatant thereof of mesenchymal stem cells. The culture medium or supernatant thereof of mesenchymal stem cells may be obtained after removing large molecules by centrifugation or filtering. In some embodiments, the culture medium or supernatant thereof of mesenchymal stem cells may be obtained by sequential filtration using a 0.45 μm filter and a 0.2 μm filter. When the culture medium of mesenchymal stem cells is a concentrate, the concentrate may be a culture of the stem cell culture medium or a supernatant thereof concentrated about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, or about 20 times.

[0095] In some embodiments, the medium composition of the present invention may be a medium composition obtained by the manufacturing method described above.

[0096] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0097] Example

[0098] Example 1. Preparation of a medium conditioned with Wharton jelly-derived mesenchymal stem cells

[0099] Example 1.1. Isolation of mesenchymal stem cells derived from Wharton jelly

[0100] Informed consent was obtained from healthy mothers who had given birth normally. Umbilical cords were collected at the time of delivery and treated with saline solution and antibiotics. All blood was removed from the cord surface, and the umbilical cord membrane and blood vessels were removed. The gelatinous material, including Wharton's jelly, was then isolated. All procedures were performed under sterile conditions.

[0101] Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (wjMSCs) were isolated from the separated Wharton's jelly.

[0102] Example 1.2. Preparation of a medium conditioned with Wharton jelly-derived mesenchymal stem cells.

[0103] Human umbilical cord Wharton jelly-derived mesenchymal stem cells (wjMSCs) isolated in Example 1.1 were cultured in a 5% CO2 incubator (Thermofisher, USA) at 37°C using xenofree complete DMEM F12 medium supplemented with human platelet lysate (HPL) (PL Bioscience, Germany), 1× penicillin-streptomycin (GIBCO, USA), and 1× Glutamax™ (GIBCO, Carlsbad, USA). wjMSCs were subcultured in complete DMEM F12 medium, and the wjMSCs of the fifth passage were seeded at 50,000 cells / cm on two types of tissue culture dishes (500 kPa and 3 GPa) according to substrate stiffness. 2 Cells were seeded at a density of 100 μm and cultured in complete DMEM F12 medium until 80% confluent.

[0104] Thereafter, for the preparation of chemically defined conditioned media (CDCM), the culture medium of the experimental group cultured according to substrate rigidity was replaced with the CDCM medium of the present invention (DMEM medium supplemented with transferrin, selenium, insulin, glutathione, phospholipids, and human albumin). For the negative control group, stem cells were cultured on basic cell culture plates and then replaced with low-glucose DMEM medium (Dulbecco's Modified Eagle Medium) (GIBCO, USA). After 24 hours, the existing culture medium in each plate was removed and fresh medium was added. The conditioned medium was collected every 3 days during 9 days of culture. The collected conditioned medium was sequentially filtered through a 0.45 μm filter and a 0.2 μm filter (Thermofisher, USA) and stored at 4°C for further testing and subsequent analysis. The medium conditioned on a 500 kPa substrate was designated as 500 kPa-CDCM, and the medium conditioned on a 3 GPa substrate was designated as 3 GPa-CDCM.

[0105] Example 2. Wound healing effect of medium conditioned with mesenchymal stem cells

[0106] Example 2.1. Fibroblast culture in conditioned medium

[0107] Human neonatal dermal fibroblasts (NDFB) cultured for the fifth passage (Pelobiotech, Germany) were seeded at 2.5 × 10 in 6-well plates. 5 cells / ml or 2600 cells / cm 2 The cells were seeded at a density of 100 μm and cultured in a 37°C incubator for 24 hours for attachment. DMEM medium supplemented with 10% FBS was used as a medium for proliferation of human neonatal dermal fibroblasts.

[0108] In order to confirm the effect of the conditioned medium of the present invention on fibroblasts, the 500 kPa-CDCM and 3 GPa-CDCM prepared in Example 1.2 were diluted to various concentrations (100%, 50%, 30%, and 10%) using the CDCM medium of the present invention. The medium conditioned with DMEM prepared in Example 1.2 was used as a negative control, and complete NDFB medium (DMEM medium supplemented with 1× glutamax™ and 10% fetal bovine serum (FBS)) was used as a positive control. The cell medium of each plate was removed and washed three times with 1× PBS, and then 3 ml of the medium assigned to each group was added (n=5).

[0109] Example 2.2. Improvement of fibroblast proliferation

[0110] The proliferation assay of fibroblasts was performed for 7 days, and the medium assigned to each group in Example 2.1 was replaced every 3 days. Day 1 was defined as 24 hours after replacing the medium assigned to each group (500 kPa-CDCM, 3 GPa-CDCM and their dilutions, negative control, and positive control). On day 7, cells were fixed with 4% paraformaldehyde, and nuclei were stained with DAPI for cell counting. Image J software (NIH, USA) was used to count DAPI-stained nuclei.

[0111] As a result of proliferation analysis, referring to Figures 1a and 1b, fibroblasts cultured with 100% 500 kPa-CDCM showed a significantly higher total cell number compared to complete NDFB medium (positive control) or DMEM CM (negative control) (p<0.05). Fibroblast proliferation tended to be significantly reduced in diluted concentrations (50%, 30%, and 10%) of 500 kPa-CDCM. Fibroblasts cultured with 100% 500 kPa-CDCM showed a significantly higher total cell number compared to fibroblasts cultured with 100% 3 GPa-CDCM (p<0.05).

[0112] Example 2.3. In vitro wound healing effect

[0113] For the in vitro wound healing assay, sterilized polydimethylsiloxane (PDMS) rubber blocks (20 mm × 6 mm × 4 mm) were firmly attached to the bottom of each well of a 6-well plate (Fig. 2a). Human neonatal dermal fibroblasts cultured for the fifth passage were seeded at a density of 50,000 cells / cm 2 Cells were seeded at a density of 10 μm and cultured until maximum confluency was reached. Fibroblasts were cultured in DMEM medium supplemented with 10% FBS at 37°C in an incubator with 5% CO2 and 95% humidity.

[0114] The rubber block was carefully removed to avoid disturbing cell alignment, creating a 20 mm × 6 mm rectangular wound area for cell migration or in vitro wound closure. The culture medium was aspirated and washed three times with 1× PBS. Then, 3 ml of conditioned medium was added to each well. Cell migration was measured using a migration measurement window from day 1 (24 h after conditioned medium treatment) and every 48 h thereafter until day 11. The distance between linear cell walls was measured using a microscope (Nikon, Japan), and the captured images were reconstructed using the stitching plugin of Image J software. All data were analyzed using SPSS statistical software (version 20.0; IBM Corp., Armonk, NY, IL). The Mann-Whitney test was used to determine statistical differences between the two groups. A p value less than 0.05 was considered statistically significant.

[0115] In vitro wound healing assay results, as shown in Figures 2b and 2c, showed that in the presence of conditioned media, fibroblasts covered the wound faster than the negative control. From day 5, fibroblasts treated with 500 kPa-CDCM or 3 GPa-CDCM exhibited shorter linear inter-wall distances than fibroblasts treated with DMEM-conditioned media (negative control) (p<0.05). In addition, fibroblasts treated with 500 kPa-CDCM exhibited a significantly higher migration speed than fibroblasts treated with 3 GPa-CDCM (p<0.05).

[0116] We found that conditioned medium with mesenchymal stem cells cultured on soft (500 kPa) substrates promoted proliferation and migration of human neonatal dermal fibroblasts better than medium conditioned with mesenchymal stem cells cultured on stiff (3 GPa) substrates. This suggests that environmental cues (i.e., substrate stiffness) may influence the regenerative capacity of cells in the production of conditioned medium, as mesenchymal stem cells are known to sense extracellular matrix stiffness using α- and β-integrin receptors through binding to ligands such as talin, paxillin, and vinculin. Consequently, it is suggested that by modulating substrate stiffness, conditioned medium containing mesenchymal stem cell secretions necessary for wound healing can be obtained.

[0117] Example 3. Skin improvement using medium conditioned with mesenchymal stem cells.

[0118] Example 3.1. Stimulation of skin cell growth

[0119] Human neonatal dermal fibroblasts (NDFB) (Pelobiotech, Germany) were cultured at 2,000 cells / cm 2 After seeding at a density of 10%, the plates were cultured in complete DMEM medium (containing 10% FBS) until 70% or more confluent. Each well was washed three times with 1× PBS to remove the culture medium, and the culture medium was replaced by adding the stem cell-conditioned medium prepared in Example 1.2 (500 kPa-CDCM, experimental group), complete DMEM medium (containing 10% FBS, positive control), or DMEM-conditioned medium (no additives, negative control) to each plate. The negative control was a conditioned medium obtained by culturing stem cells on a 500 kPa substrate, and DMEM was used as the medium. Forty-eight hours after replacing the culture medium, n=5 or more regions of interest (ROI) spots were randomly selected to compare cell numbers.

[0120] As a result, it was confirmed that the medium conditioned with mesenchymal stem cells showed an increase in cell number (i.e., skin regeneration effect) of approximately 250% or more than the complete DMEM medium containing 10% FBS, which was the positive control (Fig. 3a).

[0121] Example 3.2. Promotion of extracellular matrix (ECM) production

[0122] To confirm the effectiveness of stem cell culture as a skin aging inhibitor, the expression levels of elastin, fibronectin, and collagen III contained in the extracellular matrix (ECM) were confirmed.

[0123] Human neonatal dermal fibroblasts (NDFB) (Pelobiotech, Germany) were seeded at 2,000 cells / cm in 100 mm cell culture plates (SPL). 2 After seeding at a density of 100 μL, cells were cultured in complete DMEM medium (containing 10% FBS) until 70% or more confluent. Each well was washed three times with 1× PBS to remove the culture medium, and the culture medium was replaced by adding the same stem cell-conditioned medium as in Example 3.1 (500 kPa-CDCM, experimental group) or DMEM-conditioned medium (no additives, negative control group) to each plate. The plates with the replaced medium were irradiated with UV-C, a senescence inducer, at a concentration of 24.12 kJ for 30 minutes. Cells were harvested after 6 to 16 hours, and RNA was extracted using an RNA extraction kit. The extracted RNA was used to compare the gene expression levels of elastin, fibronectin, and collagen III via PCR.

[0124] As a result, we confirmed that cells cultured in a medium conditioned with mesenchymal stem cells showed a significant increase in the gene expression of extracellular matrix factors compared to cells cultured in a medium conditioned with DMEM, which is a negative control, under UV damage. Compared to the negative control, the expression of the elastin gene in cells cultured in the medium conditioned with stem cells increased by approximately 250% (Fig. 3b), the expression of the fibronectin gene increased by approximately 600% (Fig. 3c), and the expression of the collagen III gene increased by approximately 230% (Fig. 3d). This suggests that proteins that help improve skin elasticity, skin regeneration, and wrinkles can be excellently synthesized by the medium conditioned with the mesenchymal stem cells of the present invention.

[0125] Example 3.3. Inhibition of aging gene expression

[0126] Cells were cultured in the same manner as in Example 3.2 and irradiated with UV-C to induce cell senescence. The expression level of p16, a representative gene expressed during cell senescence, was confirmed. The expression level of the p16 gene was compared between the medium conditioned with DMEM before UV-C irradiation (DMEM, negative control) and the medium conditioned with DMEM after UV-C irradiation (UV-DMEM, positive control). As a result, the expression level of the p16 gene increased approximately 5-fold (500%) after UV-C irradiation. On the other hand, in cells cultured in a medium conditioned with stem cells (UV-CDCM), the expression of the p16 gene tended to decrease compared to before UV damage was induced (Fig. 3e). This represents a decrease in the expression level of the p16 gene of approximately 10-fold (1000%) compared to the positive control (UV-DMEM). In other words, this suggests that cell reversal is possible using the medium conditioned with the mesenchymal stem cells of the present invention.

Claims

1. A medium composition conditioned with mesenchymal stem cells obtained by a method including culturing mesenchymal stem cells on a low-rigidity substrate, A composition of a badge that exhibits wound healing, regeneration, wrinkle improvement, anti-aging or reverse-aging effects on the skin.

2. In paragraph 1, The culture of the above mesenchymal stem cells is performed in a culture medium containing one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin. Composition.

3. In paragraph 1, The above low-stiffness substrate is a substrate having a stiffness of 10 kPa to 3.5 GPa. Composition.

4. In paragraph 3, The above low-strength substrate is a substrate having a stiffness of 10 kPa to 1 GPa. Composition.

5. In paragraph 3, The above low-strength substrate is a substrate having a stiffness of 50 kPa to 1000 kPa. Composition.

6. In paragraph 1, The above-mentioned badge composition exhibits at least one of the effects of promoting skin cell proliferation, promoting elastin synthesis, promoting fibronectin synthesis, promoting collagen III synthesis, and inhibiting aging gene expression. Badge composition.

7. In paragraph 6, The above skin cells are fibroblasts. Composition.

8. In paragraph 1, The above mesenchymal stem cells are derived from human umbilical cord Wharton's jelly. Composition.

9. A cosmetic composition comprising an effective amount of a badge composition according to any one of claims 1 to 8.

10. In paragraph 9, The above cosmetic composition has a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray. Cosmetic composition.

11. A pharmaceutical composition for treating or preventing skin damage, comprising an effective amount of a badge composition according to any one of claims 1 to 8.

12. A pharmaceutical product for preventing or treating skin damage containing an effective amount of a badge composition according to any one of claims 1 to 8.

13. An external skin preparation for preventing or treating skin damage, comprising an effective amount of a composition according to any one of claims 1 to 8.

14. A method for producing a medium composition conditioned with mesenchymal stem cells, comprising the steps of culturing mesenchymal stem cells on a low-rigidity substrate and recovering a culture medium from the culture.

15. In paragraph 14, The above low-stiffness substrate is a substrate having a substrate stiffness of 10 kPa to 3.5 GPa. method.

16. In paragraph 14, The above low-stiffness substrate is a substrate having a substrate stiffness of 10 kPa to 1 GPa. method.

17. In paragraph 14, The above low-stiffness substrate is a substrate having a substrate stiffness of 50 kPa to 1000 kPa. method.

18. In paragraph 14, The above mesenchymal stem cells are isolated from human umbilical cord Wharton jelly. method.

19. In paragraph 14, The culture of the above mesenchymal stem cells is performed in a culture medium containing one or more of transferrin, selenium, insulin, glutathione, phospholipids, and human albumin. method.

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