Adipose-derived stem cells having enhanced immunomodulatory factor secretion reactivity, method for producing same, and use thereof

Abstract

The present invention relates to adipose-derived stem cells having enhanced immunomodulatory factor secretion reactivity, a method for producing same, and a use thereof. More specifically, the present invention relates to: a method for mass-culturing adipose-derived stem cells having enhanced immunomodulatory factor secretion reactivity, the method comprising a step for treating adipose-derived stem cells with a combination of lipoic acid, cobalamin, and ascorbic acid; adipose-derived stem cells cultured by the method; and a use of the stem cells in the prevention or treatment of immune diseases.

Description

Adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, method for manufacturing the same, and uses thereof

[0001] The present invention relates to adipose-derived stem cells with enhanced reactivity to the secretion of immunomodulatory factors, a method for producing the same, and uses thereof. More specifically, the present invention relates to a method for mass culture of adipose-derived stem cells with enhanced reactivity to the secretion of immunomodulatory factors, comprising the step of treating the adipose-derived stem cells with a combination of lipoic acid, cobalamin, and ascorbic acid; adipose-derived stem cells cultured by the said method; and uses of said stem cells in the prevention or treatment of immune diseases.

[0002] Stem cells are undifferentiated cells capable of differentiating into various cell types through their self-renewal and differentiation capabilities, and they are being actively researched in the fields of cell therapy and regenerative medicine. Stem cells are broadly classified into embryonic stem cells (ESCs) and adult stem cells (ASCs). Among these, adult stem cells are divided into hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs); mesenchymal stem cells are reported to possess immune-modulating capabilities and multilineage differentiation potential. Adipose-derived stem cells (ADSCs) are a form of mesenchymal stem cells isolated from adipose tissue; they have the advantage of higher yield and faster proliferation rates compared to bone marrow-derived stem cells (BMSCs). Adipose-derived stem cells can be non-invasively harvested through liposuction and are suitable for autologous transplantation, offering a low risk of immune rejection and high safety. Adipose-derived stem cells are known to secrete various growth factors and cytokines through paracrine effects, performing various physiological functions such as wound healing, anti-inflammation, and immunomodulation. In particular, they secrete anti-inflammatory and immunomodulatory factors such as Transforming Growth Factor Beta 1 (TGF-β1), Prostaglandin E2 (PGE2), and Interleukin-6 (IL-6), making them highly promising for the treatment of autoimmune and inflammatory diseases.

[0003] According to recent studies, adipose-derived stem cells are reported to alleviate autoimmune responses by regulating the activity of Natural Killer (NK) cells via Interleukin-2 (IL-2) and modulating the immune responses of Regulatory T cells (Treg) and Type 2 Helper T cells (Th2). These characteristics suggest the potential for adipose-derived stem cells to be utilized as immunomodulatory therapeutic agents that maintain immune system homeostasis and contribute to the alleviation of inflammatory responses. However, cell therapies utilizing autologous cells have several limitations. In particular, in patients with autoimmune or inflammatory diseases, the patient's own stem cells may be affected by the disease, resulting in reduced inflammatory and immune activity compared to healthy individuals. Consequently, the ability to secrete immunomodulatory factors is reduced, which is highly likely to limit the effectiveness of cell therapy. To overcome these issues, priming techniques have been widely used in existing research. This method induces the secretion of immunomodulatory factors by directly treating stem cells with inflammatory stimuli such as interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). However, continuous inflammatory stimulation can impair stem cell function and reduce long-term immunomodulatory effects. Consequently, there is a growing need for pre-conditioning technologies that enable cells to respond more sensitively to inflammatory environments, without relying on inflammatory stimuli.

[0004]

[0005] Prior art

[0006] Patent Document 1. Korean Published Patent No. 10-2019-0047966

[0007] The present invention aims to present an innovative therapeutic strategy for resolving the problem of reduced immunomodulatory function of adipose-derived stem cells observed in patients with autoimmune and inflammatory diseases by enhancing the immunomodulatory function of adipose-derived stem cells in vitro and then administering them to patients. Conventional priming methods utilize direct treatment of stem cells with inflammatory stimuli such as IFN-γ and TNF-α to induce the secretion of immunomodulatory factor proteins; however, this approach relies on continuous inflammatory stimulation and carries the risk of reduced long-term immunomodulatory capacity due to the accumulation of cellular fatigue caused by repetitive stimulation.

[0008] During research aimed at enhancing the immunomodulatory factor secretion ability of adipose-derived stem cells, the inventors discovered that pre-treating cells with a combination of lipoic acid, cobalamin, and ascorbic acid significantly increases the expression of immunomodulatory-related genes, while not leading to immediate protein secretion. In particular, it was confirmed that when pre-treated cells are exposed to inflammatory cytokines such as IL-4 and IL-13, the secretion of immunomodulatory factors such as PGE2, TGF-β1, and IL-2 increases significantly compared to untreated cells. Thus, the present invention reveals that gene expression of immunomodulatory factors is pre-activated during the pretreatment stage, but protein secretion occurs only when inflammatory stimuli are applied; this prevents unnecessary protein secretion and allows for the selective performance of immunomodulatory effects only in an actual inflammatory environment.

[0009]

[0010] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0011] The present invention aims to provide adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, a method for manufacturing the same, and uses thereof.

[0012]

[0013] According to one embodiment,

[0014] A step of treating adipose-derived stem cells with a combination of lipoic acid, cobalamin, and ascorbic acid;

[0015] A method for mass culture of adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness is disclosed.

[0016] In the present invention, the concentration of the lipoic acid may be 0.1 mM to 10 mM.

[0017] In the present invention, the concentration of the cobalamin may be 10 μM to 500 μM.

[0018] In the present invention, the concentration of the ascorbic acid may be 1 mM to 50 mM.

[0019] In the present invention, the molar ratio of lipoic acid, cobalamin, and ascorbic acid may be 1 : 0.1 : 1 to 20.

[0020] In the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0021]

[0022] According to another embodiment,

[0023] Adipose-derived stem cells produced by a mass culture method of adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness are disclosed.

[0024] In the present invention, the adipose-derived stem cells can promote the secretion of the immunomodulatory factor under an inflammatory environment.

[0025] In the present invention, the inflammatory environment may be atopic dermatitis.

[0026]

[0027] According to another embodiment,

[0028] A pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases is disclosed, comprising adipose-derived stem cells as an active ingredient by a method of mass culture of adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness.

[0029] In the present invention, the immune disease or inflammatory disease is Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary liver cirrhosis, dermatomyositis, Good-Feitzer syndrome, autoimmune meningitis, Sjögren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondylosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia. One or more may be selected from the group consisting of ulcerative colitis and graft-versus-host disease.

[0030] The present invention provides a technical solution capable of overcoming the limitations of existing immunomodulation methods by providing a method to pre-activate the immunomodulatory capacity of cells in vitro and induce them to exert immunomodulatory functions only when necessary due to inflammatory stimulation.

[0031] Figure 1 shows the change in proliferation rate of adipose-derived stem cells according to treatment with different concentrations of lipoic acid, cobalamin, and ascorbic acid.

[0032] Figure 2 shows changes in the expression of major immunomodulatory genes in adipose-derived stem cells according to combinations of concentrations of lipoic acid, cobalamin, and ascorbic acid.

[0033] Figure 3 shows the changes in the secretion of major immunomodulatory proteins in adipose-derived stem cells following treatment with an optimal concentration combination of lipoic acid, cobalamin, and ascorbic acid.

[0034] Figure 4 shows the results of profiling additional immunomodulatory factors of adipose-derived stem cells following treatment with an optimal concentration combination of lipoic acid, cobalamin, and ascorbic acid.

[0035] Hereinafter, adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness according to specific embodiments of the invention, a method for manufacturing the same, and uses thereof will be described in detail. However, this is presented as one example of the invention and does not limit the scope of the invention, and it is obvious to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention. Throughout this specification, unless otherwise specifically stated, "includes" or "contains" refers to the inclusion of any component (or constituent) without any particular limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0036] As used herein, the term "treatment" means any form of treatment or prevention that provides effects, including improvement of the individual's condition, delay of disease progression, delay of symptom onset, or slowing of symptom progression, to an individual who suffers from a disease or is at risk of developing a disease. Accordingly, the term "treatment" includes preventive treatment of the individual that prevents the onset of symptoms. Furthermore, the terms "treatment" and "prevention" are not intended to mean the cure or complete elimination of symptoms.

[0037] As used in this specification, the term "improvement" may mean any action that at least reduces parameters related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0038] As used herein, the term “object” means an animal including animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the object may be a mammal, particularly a human.

[0039]

[0040]

[0041]

[0042] 1. Mass culture method of adipose-derived stem cells

[0043] The present invention relates to a method for mass culture of adipose-derived stem cells with enhanced secretory responsiveness to immunomodulatory factors, and presents a method for mass culture of adipose-derived stem cells that promotes the selective secretion of immunomodulatory factors in an inflammatory stimulus environment. Adipose-derived stem cells are an abundant cell source possessing self-renewal and multipotential differentiation capabilities, and play an important role in tissue regeneration and the development of immunomodulatory therapeutic agents due to their excellent immunomodulatory capabilities. In particular, the present invention provides a mass culture method capable of enhancing the immunomodulatory ability of adipose-derived stem cells and maximizing the secretion of immunomodulatory factors in an inflammatory stimulus environment through pretreatment with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0044]

[0045] The present invention

[0046] A step of treating adipose-derived stem cells with a combination of lipoic acid, cobalamin, and ascorbic acid;

[0047] We aim to provide a method for mass culture of adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, including [the following].

[0048] In the method for mass culture of adipose-derived stem cells according to the present invention, the concentration of lipoic acid may be 0.1 mM to 10 mM.

[0049] In the method for mass culture of adipose-derived stem cells according to the present invention, the concentration of the cobalamin may be 10 μM to 500 μM.

[0050] In the method for mass culture of adipose-derived stem cells according to the present invention, the concentration of ascorbic acid may be 1 mM to 50 mM.

[0051] In the method for mass culture of adipose-derived stem cells according to the present invention, the molar ratio of lipoic acid, cobalamin, and ascorbic acid may be 1:0.1:1 to 20.

[0052] In the method for mass culture of adipose-derived stem cells according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0053]

[0054] 2. Adipose-derived stem cells

[0055] Adipose-derived stem cells produced by the above Item 1. adipose-derived stem cell culture method possess the characteristic of enhanced secretory responsiveness to immunomodulatory factors. It has been confirmed that these stem cells significantly increase the expression of immunomodulatory factors but do not lead to immediate protein secretion, and exhibit anti-inflammatory and immunomodulatory effects by promoting the secretion of immunomodulatory factors under inflammatory conditions.

[0056]

[0057] The present invention

[0058] We aim to provide adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, comprising adipose-derived stem cells treated with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0059] In the adipose-derived stem cells according to the present invention, the stem cells can increase the expression of an immunomodulatory factor.

[0060] In the adipose-derived stem cells according to the present invention, the secretion of the immunomodulatory factor may be promoted under an inflammatory environment. At this time, the inflammatory environment may include atopic dermatitis.

[0061] In the adipose-derived stem cells according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0062]

[0063] 3. Use of the above stem cells in the prevention or treatment of immune diseases and inflammatory diseases

[0064] Item 2 above. Adipose-derived stem cells can be used for the prevention and treatment of immune diseases and inflammatory diseases. These stem cells contribute to suppressing inflammatory responses and restoring immune balance in immune diseases such as atopic dermatitis, rheumatoid arthritis, and Crohn's disease. They can inhibit the progression of immune diseases through immunosuppressive and anti-inflammatory actions, and can prevent the initial onset of inflammatory diseases or alleviate symptoms by promoting the secretion of immune regulatory factors. Furthermore, they can contribute to regulating immune responses and maximizing therapeutic effects when used in combination with stem cell therapeutics.

[0065]

[0066] (1) Pharmaceutical composition

[0067] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of immune or inflammatory diseases comprising adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, comprising adipose-derived stem cells treated with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0068] In the pharmaceutical composition according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0069] In the pharmaceutical composition according to the present invention, the immune disease or inflammatory disease is Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary liver cirrhosis, dermatomyositis, Good-Fitzer syndrome, autoimmune meningitis, Sjögren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondylosis, thyroiditis, vasculitis, vitiligo, myxedema, One or more may be selected from the group consisting of pernicious anemia, ulcerative colitis, and graft-versus-host disease. Preferably, the immune disease or inflammatory disease may be atopic dermatitis.

[0070] In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered by oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernous injection, intrathecal injection, epidural injection, and rectal administration. When administered orally, for example, the pharmaceutical composition may be formulated as a tablet, or the active agent may be coated or protected from degradation in the stomach. Additionally, the composition may be administered by any device capable of delivering the active substance to target cells. The route of administration may vary depending on the general condition and age of the subject being treated, the nature of the treatment conditions, and the selected active ingredient.

[0071] In the pharmaceutical composition according to the present invention, the suitable dosage of the pharmaceutical composition varies depending on factors such as the formulation method, the mode of administration, the patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the pharmaceutical composition may be administered as a single or multiple doses, or divided into 1 to 4 doses per day. For example, the pharmaceutical composition may contain 0.01 mg / kg to 100 mg / kg, preferably 0.02 mg / kg to 90 mg / kg, and more preferably 0.03 mg / kg to 80 mg / kg per adult.

[0072] In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer. Furthermore, the pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant, or skin patch. Additionally, the pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans.

[0073] In the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier may be a solid or a liquid and may be one or more selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release regulators, wetting agents, stabilizers, suspending agents, and lubricants. Additionally, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0074] In one embodiment, suitable fillers may include, but are not limited to, sugars (e.g., dextrose, sucrose, maltose and lactose), starch (e.g., corn starch), sugar-alcohols (e.g., mannitol, sorbitol, maltitol, erythritol and xylitol), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose).

[0075] In one embodiment, suitable binders may include, but are not limited to, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof.

[0076] In one embodiment, suitable preservatives may include, but are not limited to, benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA, or mixtures thereof.

[0077] In one embodiment, suitable disintegrant may be sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or a mixture thereof, but is not limited thereto.

[0078] In one embodiment, suitable sweeteners may include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose, or mixtures thereof.

[0079] In one embodiment, suitable glidant may be silica, colloidal silicon dioxide, talc, etc., but is not limited thereto.

[0080] In one embodiment, suitable lubricants may include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride wax, or mixtures thereof.

[0081]

[0082] (2) Cosmetic composition

[0083] The present invention aims to provide a cosmetic composition for the prevention or improvement of immune or inflammatory diseases comprising adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, comprising adipose-derived stem cells treated with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0084] In the cosmetic composition according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0085] In a cosmetic composition according to the present invention, the immune disease or inflammatory disease is Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary liver cirrhosis, dermatomyositis, Good-Feitzer syndrome, autoimmune meningitis, Sjögren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondylosis, thyroiditis, vasculitis, vitiligo, myxedema, One or more may be selected from the group consisting of pernicious anemia, ulcerative colitis, and graft-versus-host disease. Preferably, the immune disease or inflammatory disease may be atopic dermatitis.

[0086] In the cosmetic composition according to the present invention, the cosmetic composition may additionally include a dermatologically acceptable carrier. The dermatologically acceptable carrier may include, but is not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, hygroscopic agent, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, preservative, lower alcohol, etc., and its type and concentration may vary and may include parts that a person skilled in the art can modify within the scope of the present invention.

[0087] In the cosmetic composition according to the present invention, in addition to the active ingredient of the present invention, the cosmetic composition may include functional substances as needed, such as whitening agents, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, sunscreens, antibiotics, anti-acne agents, perfumes, and dyes, and these may be included in the cosmetic composition according to the present invention in amounts commonly used in the field of cosmetics. To enhance the functional effect, the cosmetic composition of the present invention may additionally contain one or more moisturizing active ingredients exhibiting the same or similar functions.

[0088] In the cosmetic composition according to the present invention, the cosmetic composition may be prepared in the form of a general emulsion formulation and a solubilizing formulation. Cosmetics in the form of an emulsion include nourishing lotions, creams, essences, etc., and cosmetics in the form of a solubilizing formulation include softening lotions. In addition to the active ingredient of the present invention, the cosmetic composition may also be prepared in the form of an adjuvant for topical or systemic application commonly used in the art by containing a dermatologically acceptable medium or base. Suitable cosmetic formulations may be provided, for example, in the form of a solution, gel, solid or paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranulocyte, or an ionic (liposome) or non-ionic vesicular dispersant, or in the form of a cream, skin toner, lotion, powder, ointment, spray, or conceal stick. Additionally, it may be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

[0089] In the cosmetic composition according to the present invention, the cosmetic composition may be formulated into one or more selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a perfume, a pack, a softening water, a lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing product, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0090]

[0091] (3) Food composition

[0092] As used in this specification, the term “food” refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone some degree of processing to become ready for direct consumption, and in a conventional sense may include food, food additives, functional foods, and beverages.

[0093] As used in this specification, the terms “functional food” or “health functional food” refer to a group of foods to which added value has been added to the food by using physical, biochemical, or biotechnological methods to act or manifest the function of the food for a specific purpose, or to foods designed and processed to sufficiently express in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery, which are inherent in the food composition; specifically, they may be health functional foods. The functional food may include food science-acceptable food additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods. The types of health functional foods may include, but are not limited to, powder, granules, tablets, capsules, or beverage forms.

[0094] According to one embodiment,

[0095] The present invention aims to provide a food composition for the prevention or improvement of immune or inflammatory diseases comprising adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, comprising adipose-derived stem cells treated with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0096] In the food composition according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0097] In the food composition according to the present invention, the food composition may be used to improve fatigue or enhance exercise performance. For example, the food composition may be used to prevent or improve physical fatigue, muscle fatigue, muscle pain, decreased muscle function, muscle diseases caused by muscle wasting or muscle degeneration, muscle wasting, decreased explosiveness, or decreased endurance, but is not limited thereto.

[0098] In the food composition according to the present invention, the food is characterized as being meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, stamina drinks, alcoholic beverages, or vitamin complexes.

[0099] In the food composition according to the present invention, the food composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0100] In the food composition according to the present invention, the term “functional food or health functional food” refers to a group of foods to which added value is imparted by using physical, biochemical, or biotechnological methods to make the function of the food act or manifest for a specific purpose, or a food processed by designing it to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition; specifically, it may be a health functional food. The functional food may include food-scientifically acceptable food auxiliary additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0101]

[0102] (4) Feed composition

[0103] According to one embodiment,

[0104] The present invention aims to provide a feed composition for the prevention or improvement of immune or inflammatory diseases comprising adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, comprising adipose-derived stem cells treated with a combination of lipoic acid, cobalamin, and ascorbic acid.

[0105] In the feed composition according to the present invention, the immunomodulator may include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

[0106] In the feed composition according to the present invention, the feed may be powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc., but is not limited thereto.

[0107] In the feed composition according to the present invention, the feed composition may include binders, emulsifiers, preservatives, etc. added to prevent quality degradation, and the feed composition may include feed additives. To increase utility, amino acid preparations, vitamin preparations, enzyme preparations, flavoring agents, non-protein nitrogen compounds, silicate preparations, buffering agents, extractants, oligosaccharides, etc. may be added to the feed. In addition, feed mixing agents, etc. may be additionally included, but are not limited thereto.

[0108] Hereinafter, examples are presented to select optimal concentrations of lipoic acid, cobalamin, and ascorbic acid capable of enhancing the reactivity of immunomodulatory factors, and to verify whether the secretory reactivity of immunomodulatory factors is enhanced in adipose-derived stem cells in an environment simulating atopic dermatitis induced by IL-4 and IL-13 by treating each substance according to the present invention individually or in combination. The following examples are provided to facilitate a better understanding of the invention, and the scope of protection of the invention is not limited to the following examples.

[0109]

[0110] <Example>

[0111] Example 1: Evaluation of the effects of lipoic acid, cobalamin, and ascorbic acid concentrations on the proliferation of adipose-derived stem cells

[0112] This example was performed to evaluate the concentration-dependent effects of lipoic acid, cobalamin, and ascorbic acid on the proliferation of adipose-derived stem cells, and to explore the optimal concentration range that can maximize immunomodulatory functions without inhibiting cell proliferation.

[0113] First, adipose tissue was harvested via liposuction to isolate adipose-derived stem cells from the subcutaneous fat of patients with atopic dermatitis. The harvested adipose tissue was washed with physiological saline at room temperature to remove blood and impurities. Subsequently, the adipose tissue was placed in a collagenase type I solution and subjected to enzymatic digestion at 37°C for 60 minutes. After enzymatic digestion was complete, the adipose tissue was diluted with Dulbecco's Modified Eagle Medium (DMEM) and centrifuged at 1500 rpm for 5 minutes to separate the cells from the undigested tissue. The adipocyte layer was removed, and the precipitated cell mass was washed twice with Phosphate Buffered Saline (PBS). Washed cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotic (Penicillin-streptomycin), dispensed into T-75 flasks, and cultured for 24 hours in an incubator at 37°C under 5% CO₂ conditions. After 24 hours of culture, unattached suspended cells were removed, and the medium was replaced with fresh medium. Subsequently, the cells were cultured until they reached a confluent state of 80–90% or more; cells were then isolated via enzyme treatment (TrypLE™ Express Enzyme) and subjected to passaging culture. This process was repeated up to passage 5, and cells were frozen and stored at each passage stage. Afterward, cells from the appropriate passage were thawed according to the experimental purpose and used in each experiment.

[0114] Adipose-derived stem cells from Passage 5 were seeded into a 96-well plate at a density of 5,000 cells (5 × 10³ cells) per well. The cells were stabilized in culture for 24 hours to induce adhesion and early growth. Subsequently, the effects of lipoic acid, cobalamin, and ascorbic acid on the proliferation of adipose-derived stem cells were evaluated by treating them at different concentrations. Lipoic acid was applied at concentrations ranging from 0 to 20 mM, cobalamin from 0 to 1 mM, and ascorbic acid from 0 to 50 mM, and culture was continued for 24 hours after treatment. Cell proliferation rate was evaluated using CCK-8 (Cell Counting Kit-8, Cellomax). After 24 hours of culture, a CCK-8 solution equivalent to 10% of the culture volume was added to each well, reacted at 37°C for 2 hours, and then the absorbance was measured and analyzed at 450 nm.

[0115] As a result of the analysis, the cell proliferation rate in the cobalamin and ascorbic acid treatment groups tended to be maintained or increased regardless of concentration. In particular, the cell proliferation rate of ascorbic acid gradually increased as the concentration increased, and a significant increase in proliferation rate was confirmed at 10 mM or higher. On the other hand, in the lipoic acid treatment group, the cell proliferation rate was maintained at less than 5 mM, but showed a tendency to decrease sharply at 5 mM or higher. Cell proliferation was significantly inhibited at 10 mM or higher. These results suggest that lipoic acid may inhibit cell proliferation or induce toxicity at high concentrations. Therefore, it was confirmed that lipoic acid can be treated at less than 5 mM, while cobalamin and ascorbic acid can be treated up to a maximum of 1 mM and 50 mM, respectively (Fig. 1).

[0116]

[0117]

[0118] * Example 2: Analysis of Lipoic Acid, Cobalamin, and Ascorbic Acid Pretreatment and Optimal Concentration Combinations on Major Immunomodulatory Gene Expression in Adipose-Derived Stem Cells

[0119] In this embodiment, lipoic acid, cobalamin, and ascorbic acid were combined and treated at various concentrations to explore the optimal concentration combination by analyzing the gene expression of major immunomodulatory factors. The genes analyzed were COX-2, TGF-β1, CXCL1, and IL-2, and their expression levels were evaluated to confirm the potential for enhancing the immunomodulatory ability of adipose-derived stem cells.

[0120] Adipose-derived stem cells of passage 3 obtained in Example 1 were treated with different concentrations of lipoic acid (0–2 mM), cobalamin (0–100 μM), and ascorbic acid (0–20 mM), and the cells were cultured to passage 5 to obtain cell lysate samples for gene analysis, and gene expression analysis was performed using real-time polymerase chain reaction (Realtime PCR).

[0121] Analysis results showed that adipose-derived stem cells treated with lipoic acid, cobalamin, and ascorbic acid exhibited a tendency for significantly increased expression of immunomodulatory factor genes within specific concentration ranges. It was confirmed that the expression of COX-2, TGF-β1, and CXCL1 genes increased by approximately 4 to up to 20 times when treated with a combination of 1 mM lipoic acid, 100 μM cobalamin, and 1 to 20 mM ascorbic acid. Additionally, it was confirmed that the expression of the IL-2 gene increased by up to 6 times when treated with a combination of 1 to 2 mM lipoic acid, 10 to 100 μM cobalamin, and 1 to 20 mM ascorbic acid. These results confirmed that combination treatment with lipoic acid at a concentration of 1 mM, cobalamin at 100 μM, and ascorbic acid at 1 to 20 mM is the most suitable combination for maximizing the expression of immunomodulatory factors in adipose-derived stem cells (Fig. 2). These results suggest that the combination of lipoic acid, cobalamin, and ascorbic acid can promote the expression of key immunomodulatory factors in adipose-derived stem cells.

[0122]

[0123] Example 3: Evaluation of the effect of pretreatment on the secretion of major immunomodulatory proteins by adipose-derived stem cells under an inflammatory stimulus environment

[0124] This example was conducted to evaluate whether adipose-derived stem cells pretreated with lipoic acid, cobalamin, and ascorbic acid, either alone or in combination, promote the secretion of immunomodulatory factors (PGE2, TGF-β1, GRO-α, IL-2) in an environment mimicking atopic dermatitis induced by inflammatory stimuli (IL-4, IL-13). To this end, the protein secretion amounts of each immunomodulatory factor were quantitatively measured using an enzyme immunoassay (ELISA).

[0125] Adipose-derived stem cells from passage 1 obtained in Example 1 were treated individually or in combination with the optimal concentrations of each substance derived in Example 2 (lipoic acid 1 mM, cobalamin 100 μM, ascorbic acid 20 mM) and cultured to passage 5. Cells were placed in a 12-well plate at a density of 50,000 cells per well (5 × 10⁶). 4 Cells were aliquoted. Cells were stabilized in DMEM medium containing 10% FBS for 24 hours to induce cell adhesion and initial growth. Subsequently, to simulate the environment of atopic dermatitis, inflammatory cytokines IL-4 and IL-13 were added to the culture medium at a concentration of 1 μg / mL, respectively. After 24 hours of treatment, the secretion of immunomodulatory factor proteins in the culture medium was quantified using human PGE2 (R&D systems, KGE004B), TGF-β1 (R&D systems, DY240), GRO-α (R&D systems, DGR00B), and IL-2 (R&D systems, D2050) ELISA kits (Fig. 3).

[0126] As a result of the analysis, PGE2 secretion levels were similar in the general culture environment (Control), with 223.82, 278.10, 214.99, 214.39, and 229.85 pg / mg in the control group (Non-treated), the individual treatment groups of Lipoic acid, Cobalamin, and Ascorbic acid, and the combination treatment group (L+C+A Combination), respectively. However, in the atopic dermatitis environment (+IL-4 / IL-13), the levels were 544.69, 845.67, 651.48, 554.62, and 1466.21 pg / mg, respectively, and it was confirmed that the combination treatment group increased by approximately 2.69 times compared to the control group. In the case of TGF-β1, in a general culture environment, the values ​​were measured at 73.36, 77.21, 84.79, 99.56, and 114.04 pg / mg in the control group, individual treatment group, and combination treatment group, respectively, while in an atopic dermatitis environment, they were 81.26, 86.91, 89.67, 173.67, and 182.37 pg / mg, showing a 2.24-fold increase in the combination treatment group compared to the control group. GRO-α secretion levels were observed at 8.01, 5.36, 5.36, 6.65, and 8.37 pg / mg in the control group, individual treatment group, and combination treatment group, respectively, in a general culture environment. In the atopic dermatitis environment, values ​​were measured at 169.42, 180.43, 101.34, 218.64, and 418.69 pg / mg, showing an increase of approximately 2.47 times in the combination treatment group compared to the control group. In the case of IL-2, in the general culture environment, values ​​were measured at 7.26, 7.26, 6.01, 11.80, and 8.60 pg / mg in the control group, individual treatment group, and combination treatment group, respectively. In the atopic dermatitis environment, values ​​were 65.05, 59.21, 122.22, 78.34, and 176.87 pg / mg, showing an increase of approximately 2.71 times in the combination treatment group compared to the control group.

[0127] In conclusion, it was confirmed that the secretion of major immunomodulators such as PGE2, TGF-β1, GRO-α, and IL-2 significantly increased in an atopic dermatitis environment treated with IL-4 and IL-13. In particular, the effect was most pronounced when lipoic acid, cobalamin, and ascorbic acid were combined. It is noteworthy that even with treatment with lipoic acid, cobalamin, and ascorbic acid, the secretion of immunomodulators did not significantly increase in a standard culture environment. While PGE2, TGF-β1, GRO-α, and IL-2 all showed no significant difference compared to the control group under standard culture conditions, their secretion increased rapidly in an atopic dermatitis environment induced by IL-4 and IL-13. This implies that the combined treatment with lipoic acid, cobalamin, and ascorbic acid pre-conditioned the adipose-derived stem cells to respond more sensitively to the inflammatory environment. In other words, it was found that the body is regulated to secrete large amounts of immunomodulatory factors upon external inflammatory stimulation, preventing unnecessary activation while inducing a strong immune response in inflammatory situations.

[0128]

[0129] Example 4: Analysis of additional immunomodulatory factor expression profiles of adipose-derived stem cells under inflammatory stimulation conditions

[0130] In this example, we aimed to identify additional immunomodulatory factors secreted in response to inflammatory stimuli (IL-4, IL-13) in adipose-derived stem cells pretreated with lipoic acid, cobalamin, and ascorbic acid, in addition to PGE2, TGF-β1, GRO-α, and IL-2. To this end, cytokine array (R&D, ARY005B) analysis was performed using the culture medium of the control group and the combination treatment group obtained in Example 3.

[0131] Analysis results showed that adipose-derived stem cells pretreated with lipoic acid, cobalamin, and ascorbic acid exhibited a significant increase in the secretion of immunomodulatory factors such as IL-10, IL-1ra, IL-27, IL-32α, and Serpin E1 in an atopic dermatitis-mimicking environment treated with IL-4 and IL-13. These immunomodulatory factors are known to perform anti-inflammatory and immunosuppressive functions that suppress inflammatory responses and promote tissue recovery. In the general culture environment (Control), there was no significant difference in the expression levels of immunomodulatory factors between the control group and the group treated with the combination of lipoic acid, cobalamin, and ascorbic acid, with most being negligible or almost undetectable. However, in the atopic dermatitis environment treated with IL-4 and IL-13, a distinct increase in the secretion of various immunomodulatory factors was observed in the combination treatment group compared to the untreated control group (Fig. 4). Specifically, IL-10 levels in the combination-treated group showed almost no difference compared to the control group, at a level of 0.9 times under a normal culture environment, but increased significantly to 13.33 times under an inflammatory environment. IL-1ra was confirmed to increase 7.05 times, IL-32α 10.87 times, and Serpin E1 / PAI-1 9.38 times under an inflammatory environment. In particular, the increased expression of anti-inflammatory factors such as IL-10 and IL-1ra is interpreted as an important indicator that further enhances the potential of adipose-derived stem cell-based therapy in the treatment of inflammatory diseases such as atopic dermatitis. As in Example 3, adipose-derived stem cells pretreated with a combination of lipoic acid, cobalamin, and ascorbic acid did not induce immediate expression of immunomodulatory factors under a normal culture environment. However, under conditions where inflammatory stimuli such as IL-4 and IL-13 were applied, they exhibited a characteristic of significantly increased secretion of immunomodulatory factors. These results suggest that combined treatment with lipoic acid, cobalamin, and ascorbic acid is effective in enhancing the immunomodulatory function of adipose-derived stem cells and contributes to preparing the cells to respond selectively to inflammatory stimuli.In conclusion, pretreatment with a combination of lipoic acid, cobalamin, and ascorbic acid is evaluated as an effective strategy to maximize the immunomodulatory potential of adipose-derived stem cells and provides important experimental evidence for the development of cell therapies for the treatment of inflammatory diseases.

[0132]

[0133] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A step of treating adipose-derived stem cells with a combination of lipoic acid, cobalamin, and ascorbic acid; A method for mass culture of adipose-derived stem cells with enhanced immunomodulatory factor secretion responsiveness, including 2. In Paragraph 1, A method for mass culture of adipose-derived stem cells, characterized in that the concentration of the lipoic acid is 0.1 mM to 10 mM.

3. In Paragraph 1, A method for mass culture of adipose-derived stem cells, characterized in that the concentration of the above-mentioned cobalamin is 10 μM to 500 μM.

4. In Paragraph 1, A method for mass culture of adipose-derived stem cells, characterized in that the concentration of the ascorbic acid is 1 mM to 50 mM.

5. In Paragraph 1, A method for mass culture of adipose-derived stem cells, characterized in that the molar ratio of the lipoic acid, cobalamin, and ascorbic acid is 1:0.1:1 to 20.

6. In Paragraph 1, A method for mass culture of adipose-derived stem cells, characterized in that the above-mentioned immunomodulators include PGE2 (Prostaglandin E2), COX-2 (Cyclooxygenase-2), TGF-β1 (Transforming Growth Factor Beta 1), CXCL1 (CXC Motif Chemokine Ligand 1), GRO-α (Growth-Regulated Oncogene Alpha), IL-2 (Interleukin-2), IL-10 (Interleukin-10), IL-1ra (Interleukin-1 Receptor Antagonist), IL-27 (Interleukin-27), IL-32α (Interleukin-32 Alpha), Serpin E1 (Serine Protease Inhibitor E1), or a combination thereof.

7. Adipose-derived stem cells produced by a method according to any one of paragraphs 1 to 6, The above-mentioned adipose-derived stem cells are adipose-derived stem cells that promote the secretion of the above-mentioned immunomodulatory factor under an inflammatory environment.

8. In Paragraph 7, Adipose-derived stem cells characterized in that the above-mentioned inflammatory environment is atopic dermatitis.

9. A pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising adipose-derived stem cells produced by a method according to any one of claims 1 to 6 as an active ingredient.

10. In Paragraph 9, The above-mentioned immune or inflammatory diseases include Behcet's disease, polymyositis, dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, Good-Feitzer syndrome, autoimmune meningitis, Sjögren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondylosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative A pharmaceutical composition characterized by being selected from the group consisting of colitis and graft-versus-host disease, and at least one.

Images