Composition for treating skin wounds or scars, containing mesenchymal stem cell spheres

Abstract

The present invention provides a pharmaceutical composition for treating skin wounds or scars, which contains a therapeutically effective amount of mesenchymal stem cell spheres that are locally applied to a target site or are administered by subcutaneous or intradermal injection, according to the present invention, the mesenchymal stem cell sphere is administered to a wound or a scar site, such that skin regeneration is promoted by inducing dermis and subcutaneous recovery in the skin, and thus scar healing and recovery and skin volume maintenance can be promoted.

Description

Technical Field

[0001] This invention relates to compositions for treating skin wounds or scars, and more specifically, to pharmaceutical compositions or filler compositions for treating skin wounds or scars comprising mesenchymal stem cell spheres, which are topically coated or administered via subcutaneous or intradermal injection, as an active ingredient. Background Technology

[0002] The skin is the body's first line of defense against physical and environmental attacks. When the skin is damaged, the wound can activate the wound healing process, leading to scarring. Ideally, the wound healing process should result in skin with almost no scarring, but some individuals may experience excessive fibrosis or atrophy, resulting in keloids, hypertrophic scars, or atrophic scars. Unlike keloids and hypertrophic scars, which involve tissue hyperplasia, atrophic scars form depressed scars. The depressed topography of atrophic scars is thought to be due to inadequate compensation of dermal collagen and connective tissue after injury. Atrophic scars can occur due to a variety of causes, including scleroderma, stretch marks during pregnancy, acne, surgery, and accidents. Pathological scars, especially when located in visible areas of the body, can significantly reduce quality of life and induce psychological stress. This invention provides a novel treatment method utilizing self-assembled adipose-derived mesenchymal stem cells (MSCs) that can help improve quality of life in scar treatment.

[0003] Various treatments have been used to improve the appearance of scars. These include incisions, laser therapy, and dermal filler injections. Commonly used dermal fillers include hyaluronic acid (HA), collagen, poly-L-lactic acid, and calcium hydroxylapatite. However, hyaluronic acid and collagen are fillers with short-term effects, lasting only a few months. Conversely, permanent fillers such as polymethyl methacrylate (PMMA) and silicone, while offering longer-lasting results, carry a higher risk of complications such as granulomas, silicone embolism syndrome, and nodule formation.

[0004] To overcome the limitations of artificial fillers, new approaches are needed, and the use of stem cells is emerging as a new alternative. Mesenchymal stem cells (MSCs), as adult stem cells, have been shown to exhibit excellent potential in promoting wound healing due to their self-regenerative capacity, secretion of regeneration-promoting factors, and immunomodulatory abilities. They can be obtained from various tissues such as bone marrow and adipose tissue. Currently, most cell therapy agents are administered via injection or transplantation in single-cell form. Single-cell MSCs have low survival rates, and transplant survival rates are extremely low. Therefore, when using MSCs as skin regeneration therapy agents, it is necessary to improve post-transplant survival rates and increase transplant survival rates, especially for the treatment of atrophic scars caused by connective tissue loss; developing a cell therapy agent rich in connective tissue components is crucial.

[0005] To address the aforementioned problems, the inventors of this invention established optimal physical culture conditions for skin regeneration and created a composition containing stem cells rich in extracellular matrix that can be transplanted and maintain long-term volume, thus completing this invention. Furthermore, the inventors manufactured mesenchymal stem cell spheroids, thereby confirming that MSCs transplanted into the skin maintain skin volume long-term. This demonstrates that mesenchymal stem cell spheroids overcome the limitations of existing cell-based therapies and, as skin fillers, have a more sustained effect. Summary of the Invention

[0006] The present invention aims to provide a pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheres, which are administered by topical application to a target site or by subcutaneous or intradermal injection.

[0007] In addition, the present invention provides a pharmaceutical composition for treating skin wounds or scars comprising mesenchymal stem cell spheres and a pharmaceutically acceptable carrier.

[0008] In addition, the present invention provides a filler composition for skin regeneration comprising mesenchymal stem cell spheres.

[0009] In addition, the present invention provides the use of mesenchymal stem cell spheres according to the invention for manufacturing a medicament for treating skin wounds or scars that is topically applied to a target site or administered via subcutaneous or intradermal injection.

[0010] In addition, the present invention provides mesenchymal stem cell spheres that can be locally coated on a target site or administered via subcutaneous or intradermal injection for the treatment of skin wounds or scars.

[0011] In addition, the present invention provides a method for treating skin wounds or scars by topically applying or administering a therapeutically effective amount of mesenchymal stem cell spheres to a target site in a subject who requires treatment of skin wounds or scars via subcutaneous or intradermal injection.

[0012] Unless otherwise defined, all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art. It should also be understood that terms such as those defined in commonly used dictionaries should be interpreted as having the same meaning as they have in the context of the relevant technical field, and should not be interpreted as having an idealized or overly formal meaning unless expressly defined as such in this application.

[0013] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting. When used in this application, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, when the terms “comprising,” “including,” “having,” “with,” or variations thereof are used in any part of the detailed description and / or claims, the terminology as described above is intended to be general in a manner similar to the term “comprising.”

[0014] Unless otherwise stated in this application, descriptions of numerical ranges are used herein only as a way of abbreviating the individual values ​​to which each range belongs, and each individual value is incorporated in the specification as it is individually mentioned in this application. The use of any examples or representative language (e.g., "as shown") provided in this application is solely intended to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. The language in the specification should not be construed as indicating that any unclaimed element is essential for the implementation of the invention.

[0015] The inventors of this invention have confirmed that mesenchymal stem cell spheroids produced by three-dimensionally culturing adipose tissue-derived mesenchymal stem cells exhibit excellent expression levels or production capacity of Type I collagen (ColI), fibronectin (FN1), hyaluronic acid (HA), glycosaminoglycan (GAG), tenascin (TNC), and proteoglycan-4 (PRG4). Therefore, the mesenchymal stem cell spheroids according to this invention have excellent effects in skin regeneration, treatment of skin wounds and scars, and in particular, the optimal dosage and method of administration for therapeutic efficacy have been confirmed.

[0016] This invention relates to a pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheres, which are administered by topical application to a target site or by subcutaneous or intradermal injection.

[0017] Mesenchymal stem cell spheroids

[0018] The mesenchymal stem cell spheres of the present invention can be manufactured by two-dimensional culture of mesenchymal stem cells (hMSCs) and using three-dimensional culture technology.

[0019] In one embodiment, the mesenchymal stem cell spheroids of the present invention (also referred to as MiB in this specification) can be manufactured by isolating mesenchymal stem cells (hMSCs) from human adipose tissue, culturing them in two dimensions, and using three-dimensional culture technology.

[0020] The aforementioned "two-dimensional culture" refers to the monolayer culture of cells in all types of culture containers, such as cell culture flasks or flat culture dishes.

[0021] The aforementioned "three-dimensional culture" involves the simultaneous interaction between the cell and the three-dimensional whole surrounding it, and it has the characteristic that the cell can grow in all directions.

[0022] The aforementioned "mesenchymal stem cell spheres (MiB)" refer to unit spheres of various sizes, which are the self-assembly form of cells.

[0023] In this invention, the aforementioned mesenchymal stem cell spheroids are three-dimensional cell aggregates of mesenchymal stem cells produced by culturing mesenchymal stem cells. This means that the cells do not dissociate but aggregate together in a three-dimensional cellular tissue morphology. More preferably, they are three-dimensional cell aggregates of adipose-derived mesenchymal stem cells.

[0024] The aforementioned mesenchymal stem cell spheroids can be obtained by culturing adult stem cells, which can be either commercially available stem cells or stem cells isolated from living tissue.

[0025] The aforementioned mesenchymal stem cell spheroids can effectively induce skin regeneration and the healing of skin wounds and scars. As mentioned above, the expression of growth factors or collagen production related to skin regeneration and the healing of skin wounds and scars induced by mesenchymal stem cell spheroid treatment is significantly higher than that MSC single cell treatment.

[0026] In this invention, the diameter of the mesenchymal stem cell spheres can be 10 to 800 μm, preferably 20 to 600 μm, more preferably 30 to 400 μm, even more preferably 60 to 300 μm, and even more preferably 80 to 200 μm.

[0027] In this invention, the number of mesenchymal stem cells contained in one of the above-mentioned mesenchymal stem cell spheres can be 200 to 800 cells, preferably 250 to 750 cells, more preferably 300 to 700 cells, and even more preferably 400 to 600 mesenchymal stem cells.

[0028] In this invention, the wet weight of the aforementioned mesenchymal stem cell spheroids can be 1 to 6 μg, preferably 2 to 5 μg, more preferably 2.5 to 4 μg, and even more preferably 3.19 ± 0.5 μg. The aforementioned wet weight of the mesenchymal stem cell spheroids refers to the wet weight of one mesenchymal stem cell spheroid.

[0029] In this invention, the dry weight of the aforementioned mesenchymal stem cell spheroids can be 250 to 500 ng, preferably 280 to 450 ng, more preferably 300 to 400 ng, and even more preferably 303 to 370 ng. The dry weight of the aforementioned mesenchymal stem cell spheroids refers to the dry weight of one mesenchymal stem cell spheroid.

[0030] In this invention, the aforementioned mesenchymal stem cells can be any one or more selected from adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and peripheral blood-derived mesenchymal stem cells. Preferably, they can be adipose-derived mesenchymal stem cells, and more preferably, they can be human adipose-derived mesenchymal stem cells.

[0031] In this invention, the aforementioned mesenchymal stem cell spheroids may comprise mesenchymal stem cells (MSCs) and extracellular matrix (ECM). Specifically, the extracellular matrix may comprise collagen, fibronectin, hyaluronic acid (HA), elastin (EL), or glycosaminoglycan (GAG), etc.

[0032] In addition, the collagen mentioned above is preferably type 1 collagen.

[0033] More specifically, the mesenchymal stem cell spheroids according to the present invention may have higher expression of collagen, fibronectin, hyaluronic acid (HA), elastin (EL), or glycosaminoglycan (GAG) compared to the expression of a single mesenchymal stem cell or dermal fibroblast.

[0034] In this invention, the mesenchymal stem cell spheroids mentioned above may be selected from fibroblast growth factor 2 (FGF2), platelet-derived growth factor subunit A (PDGFA), vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1) with increased expression of any one or more of these factors.

[0035] Preferably, the mesenchymal stem cell spheroids mentioned above may be characterized by increased expression of vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), or both.

[0036] In addition, preferably, the mesenchymal stem cell spheroids mentioned above can be maintained by the expression of fibroblast growth factor 2 (FGF2) or platelet-derived growth factor ablation factor (PDGFA).

[0037] More specifically, the mesenchymal stem cell spheroids according to the present invention may have a higher expression of any one or more of vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1) compared to the expression of mesenchymal stem cells or dermal fibroblasts.

[0038] Furthermore, the mesenchymal stem cell spheres according to the present invention may express fibroblast growth factor 2 (FGF2) or platelet-derived growth factor ubunit A (PDGFA) at the same level as the expression of mesenchymal stem cells or dermal fibroblasts.

[0039] In this invention, the mesenchymal stem cell spheroids are positive for one or more biomarkers selected from CD29, CD44, CD73, CD90 and CD105, and negative for one or more biomarkers selected from CD31, CD45, CD79a, CD117 and HLA-DR.

[0040] The mesenchymal stem cell spheroids contain cells that are positive for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any one or more markers selected from CD29, CD44, CD73, CD90, and CD105. In addition, the mesenchymal stem cell spheroids contain cells that are negative for at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, or 2% of any one or more markers selected from CD31, CD45, CD79a, CD117, and HLA-DR.

[0041] More specifically, the mesenchymal stem cell spheroids contain cells that positively express at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of CD29, CD44, CD73, CD90, and CD105. Furthermore, the mesenchymal stem cell spheroids contain cells that negatively express at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, or 2% of CD31, CD45, CD79a, CD117, and HLA-DR.

[0042] In addition, the mesenchymal stem cell spheroids contain cells that positively express at least 95%, 96%, 97%, 98%, or 99% of CD29, CD44, CD73, CD90, and CD105. Furthermore, the mesenchymal stem cell spheroids contain cells that negatively express at least 2%, 1.5%, 1%, or less of CD31, CD45, CD79a, CD117, and HLA-DR.

[0043] The mesenchymal stem cell spheroids of the present invention, being derived from adipose tissue, exhibit positive expression of CD29, CD44, CD73, CD90, and CD105 markers, and negative expression of CD31, CD45, CD79a, CD117, and HLA-DR markers.

[0044] The mesenchymal stem cell spheroids of the present invention can express one or more proteins selected from tenascin (TNC), fibronectin (FN1), proteoglycan-4 (PRG4), COL5A3 (Collagen Type V Alpha3 Chain), COL7A1 (Collagen Type VII Alpha 1 Chain), EVPL (Envoplakin), EFEMP2 (Fibulin-4), MFAP2 (Microfibril Associated Protein 2), FBLN2 (Fibulin-2), HSPG2, FBLN1 (Fibulin-1), and COL6A3 (Collagen alpha-3 (VI) chain).

[0045] Specifically, the mesenchymal stem cell spheroids of the present invention have significantly higher expression levels of tenascin (TNC) and fibronectin (FN1) compared to individual mesenchymal stem cells.

[0046] In addition, the mesenchymal stem cell spheroid-specific expression proteoglycan-4 (PRG4) of the present invention can re-express proteins such as COL5A3 (Collagen Type V Alpha 3 Chain), COL7A1 (Collagen Type VII Alpha 1 Chain), EVPL (Envoplakin), EFEMP2 (Fibulin-4), and MFAP2 (Microfibril Associated Protein 2) compared with single cells.

[0047] The increased expression level mentioned above can, for example, refer to an increase in the level of any of the proteins mentioned above, or their genes, by at least 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to typical mesenchymal stem cells.

[0048] In some embodiments, the mesenchymal stem cell spheres used in the treatment may also be named TRTP-101.

[0049] Composition for wound or scar treatment containing mesenchymal stem cell spheres

[0050] The present invention provides a pharmaceutical composition or cell therapy composition for treating skin wounds or scars containing a therapeutically effective amount of mesenchymal stem cell spheres.

[0051] The above-mentioned pharmaceutical composition can be applied topically or administered subcutaneously or intradermally to areas requiring skin regeneration, specifically the restoration of the entire skin layer down to the dermis or subcutaneous layer, thereby treating skin wounds or scars.

[0052] In this invention, the above-mentioned pharmaceutical composition can be formulated into various dosage forms such as injection, infusion, spray, liquid, suspension, external application, or patch, and preferably into injection, infusion, suspension, or external application.

[0053] The term "treatment" as used above refers to all actions that improve or advantageously alter the symptoms of the aforementioned disease through administration of mesenchymal stem cell spheres according to the present invention. For example, compared to natural healing, it may provide skin regeneration or wound or scar healing within a shortened time. The aforementioned treatment may include improvement and / or relief of wounds or scars. Improvement of wounds or scars refers to reducing the severity of wounds or scars. Furthermore, the aforementioned treatment may include both treating wounds and / or wound-related diseases. The aforementioned treatment may refer to the healing and / or regeneration of damaged tissue induced by a wound. The aforementioned wound treatment may refer to skin regeneration, specifically, the restoration of the dermis within the skin. Furthermore, the aforementioned treatment may maintain the original composition of the aforementioned damaged tissue. Moreover, the aforementioned treatment may minimize complications and / or scarring associated with wound-related diseases while promoting the healing and / or regeneration of the aforementioned damaged tissue.

[0054] In addition, the present invention provides a pharmaceutical composition for skin regeneration, skin wound or scar treatment comprising mesenchymal stem cell spheres and a pharmaceutically acceptable carrier.

[0055] The aforementioned pharmaceutical compositions for skin regeneration, skin wound and scar treatment may include pharmaceutically acceptable conventional inactive carriers or diluents. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical compositions of the present invention include: excipients, such as starch, sugar and mannitol; fillers and extenders, such as calcium phosphate; cellulose derivatives, such as carboxymethyl cellulose, hydroxypropyl cellulose; binders, such as gelatin, alginate and polyvinylpyrrolidone; lubricants, such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol; disintegrants, such as povidone, crospovidone; surfactants, such as polysorbate, cetyl alcohol and glycerin, but are not limited thereto. The aforementioned pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subjects receiving their transplants. As diluents, there are no limitations, but examples include saline, water-soluble buffers, solvents, suspending agents and / or dispersing agents. In addition, for example, injectable preparations may further include preservatives, analgesics, solubilizers, suspending agents, or stabilizers, while topical preparations may further include bases, excipients, lubricants, suspending agents, or preservatives.Additionally, in one embodiment, the suspending agents further include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM / F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, AminoMax±complete medium, EBM (Endothelial Basal Medium), Chang's Medium, MesenCult-XF, and DMEM / HG (Dulbecco's Modified Eagle's Medium high-performance medium). Glucose-rich medium (Dulbecco's Modified Eagle's Medium - High Glucose) or MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium - Low Glucose, MCDB+Dulbecco's Modified Eagle's Medium - Low Glucose), etc., but not limited thereto. The compositions of this invention can be used without freezing, or can be frozen for later use. If freezing is necessary, a standard cryopreservative (e.g., DMSO, glycerol, Epilife) can be added to the cell population before freezing. ®Cell freezing culture medium (Cascade Biologics). Additionally, in one embodiment, the topical drug delivery formulation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, medicated bandage, emulsion, or a combination thereof. The above-mentioned topical drug delivery formulation may, as needed, appropriately incorporate ingredients used in topical drug delivery formulations of conventional pharmaceuticals, such as aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients. The above-mentioned topical preparations may also be appropriately mixed with metal masking agents such as disodium EDTA, trisodium EDTA, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, as well as caffeine, tannins, verapamil, licorice extract, glycyrrhizin, hot water extract of calin fruit, various herbs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, trehalose, and other sugars.

[0056] The mixing ratio of the pharmaceutical composition of a specific embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional ingredients as described above.

[0057] Administration methods and dosage of mesenchymal stem cell spheroids for the treatment of skin wounds or scars.

[0058] This invention provides a method for treating skin wounds or scars by topically applying or administering a therapeutically effective amount of mesenchymal stem cell spheres via subcutaneous or intradermal injection to a target site in a subject requiring treatment of skin wounds or scars.

[0059] The mesenchymal stem cell spheres of the present invention can treat skin regeneration, skin wounds or scars by administering a therapeutically effective dose to a subject who requires treatment of skin wounds or scars.

[0060] The mesenchymal stem cell spheroids of the present invention, or pharmaceutical compositions comprising them, can be administered indirectly or directly using methods of administration commonly used in the art. In embodiments of the present invention, the aforementioned mesenchymal stem cell spheroids can be administered subcutaneously, transdermally (by coating), or intradermally. Furthermore, the administration can be direct to the target site where skin regeneration through collagen production is required for the treatment of skin wounds or scars.

[0061] In this invention, the target area may include damaged or depressed portions of the cell layers constituting the skin, such as the epidermis, dermis, and subcutaneous layer. Preferably, the target area may include damaged or depressed portions of the dermis, or it may include damaged or depressed portions of the subcutaneous layer. Specifically, dermal damage or depression can refer to damage or depression from the epidermis to the dermis, while subcutaneous damage or depression can refer to damage or depression of the entire skin layer, including the epidermis, dermis, and subcutaneous layer.

[0062] The above-mentioned coating is a transdermal drug delivery method, which refers to all methods of bringing the above-mentioned mesenchymal stem cell spheres or the drug composition containing them into contact with the target site by appropriate means, thereby allowing the above-mentioned composition to be absorbed into the skin.

[0063] In this invention, the above-mentioned mesenchymal stem cell spheres can be administered once or multiple times.

[0064] In this invention, the aforementioned mesenchymal stem cell spheroids can be administered once, twice, three times, four times, five times, or more. Preferably, they can be administered once, twice, or three times. More preferably, they can be administered once. Even with a single administration, the mesenchymal stem cell spheroids of this invention have therapeutic effects on skin regeneration, skin wounds, or scars.

[0065] The aforementioned "therapeuticly effective amount" refers to an amount sufficient to demonstrate a therapeutic effect when administered to a subject requiring treatment for skin regeneration or skin wounds or scars.

[0066] In the dosage administered to the subject, the unit dosage may contain 1 to 2 × 10⁻⁶ units. 5 The number of mesenchymal stem cell spheroids mentioned above can be, for example, a lower limit of one or more, or two or more, and an upper limit of 2 × 103. 5 Less than 1.8 × 10 5 Less than 1.6 × 10 5 Less than 1.4 × 10 5 Less than 1.2 × 10 5 Less than one, 1×10 5 Less than 1, 9.5 × 10 4 Less than 1, 9×10 4 Less than 1, 8.5 × 10 4 Less than 1, 8×10 4 Less than 1, 7.5 × 10 4 Less than 1, 7×10 4 Less than 1, 6.5 × 10 4 Less than 1, 6×10 4 Less than 5.5 × 104 Less than 1, 5×10 4 Less than 1, 4.5 × 10 4 Less than 1, 4×10 4 Less than 1, 3.5 × 10 4 Less than 1, 3×10 4 Less than 1, 2.5 × 10 4 Less than 1, 2×10 4 Less than 1.5 × 10 4 Less than one, 1×10 4 Less than 1, 9.5 × 10 3 Less than 1, 9×10 3 Less than 1, 8.5 × 10 3 Less than 1, 8×10 3 Less than 1, 7.5 × 10 3 Less than 1, 7×10 3 Less than 1, 6.5 × 10 3 Less than 1, 6×10 3 Less than 5.5 × 10 3 Less than 1, 5×10 3 Less than 1, 4.5 × 10 3 Less than 1, 4×10 3 Less than 1, 3.5 × 10 3 Less than 1, 3×10 3 Less than 1, 2.5 × 10 3 Less than one, or 2 × 10 3 The number of mesenchymal stem cells contained in one of the above-mentioned mesenchymal stem cell spheres may be 200 to 800 cells, preferably 250 to 750 cells, more preferably 300 to 700 cells, and even more preferably 400 to 600 cells.

[0067] Specifically, the number of mesenchymal stem cells contained in a unit dosage can be 1 × 10⁻⁶. 2 8 × 10 cells 7 For example, the lower limit could be 1.0 × 10⁶ cells. 2 More than one cell, 1.2 × 10 2 More than one cell, 1.4 × 10 2 More than one cell, 1.6 × 10 2 More than one cell, 1.8 × 10 2 More than one cell, 2.0 × 10 2 More than one cell, 2.2 × 10 2 More than 10 cells, 2.4 × 10 2 More than one cell, 2.6 × 10 2More than one cell, 2.8 × 10 2 More than one cell, 3.0 × 10 2 More than 10 cells, 3.2 × 10 2 More than one cell, 3.4 × 10 2 More than one cell, 3.6 × 10 2 More than one cell, 3.8 × 10 2 More than 10 cells, 4.0 × 10 2 More than 10 cells, 4.2 × 10 2 More than 10 cells, 4.4 × 10 2 More than one cell, 4.6 × 10 2 More than one cell, 4.8 × 10 2 More than one cell, 5.0 × 10 2 More than one cell, 6.0 × 10 2 More than one cell, 8.0 × 10 2 More than one cell, 1.0 × 10 3 More than one cell, 1.2 × 10 3 More than one cell, 1.4 × 10 3 More than one cell, 1.6 × 10 3 More than one cell, 1.8 × 10 3 More than one cell, 2.0 × 10 3 More than one cell, 2.2 × 10 3 More than 10 cells, 2.4 × 10 3 More than one cell, 2.6 × 10 3 More than one cell, 2.8 × 10 3 More than one cell, 3.0 × 10 3 More than 10 cells, 3.2 × 10 3 More than one cell, 3.4 × 10 3 More than one cell, 3.6 × 10 3 More than one cell, or 3.8 × 10 3 More than one cell, with an upper limit of 8×10. 7 Less than 1 cell, 8.6 × 10 7 Less than 1 cell, 8.4 × 10 7 Less than 1 cell, 8.2 × 10 7 Less than one cell, 8×10 7 Less than 1 cell, 7.8 × 10 7 Less than 1 cell, 7.6 × 10 7 Less than 1 cell, 7.4 × 10 7 Less than 1 cell, 7.2 × 10 7 Less than one cell, 7×10 7 Less than 1 cell, 6.8 × 10 7Less than 1 cell, 6.6 × 10 7 Less than 1 cell, 6.4 × 10 7 Less than 1 cell, 6.2 × 10 7 Less than one cell, 6×10 7 Less than 1 cell, 5.5 × 10 7 Less than one cell, 5×10 7 Less than 1 cell, 4.5 × 10 7 Less than one cell, 4×10 7 Less than 1 cell, 3.5 × 10 7 Less than one cell, 3×10 7 Less than 1 cell, 2.5 × 10 7 Less than one cell, 2×10 7 Less than 1 cell, 1.5 × 10 7 Less than one cell, 1×10 7 Less than one cell, 9×10 6 Less than one cell, 8×10 6 Less than one cell, 7×10 6 Less than one cell, 6×10 6 Less than one cell, 5×10 6 Less than one cell, 4×10 6 Less than one cell, 3×10 6 Less than 10 cells, 2.0 × 10 6 1 cell, or 1.5 × 10 6 Less than one cell.

[0068] In this invention, the unit dosage can be 50uL, 60uL, 70uL, 80uL, 90uL, 100uL, 125uL, 150uL, 175uL, 200uL, 225uL, 250uL, 275uL, 300uL, 325uL, 350uL, 375uL, 400uL, 425uL, 450uL, 475uL, or 500uL.

[0069] In one embodiment, the dosage per volume unit at the target site may be 1 to 2 × 10⁻⁶ units. 5 The number of mesenchymal stem cell spheroids mentioned above can be, for example, a lower limit of one or more, or two or more, and an upper limit of 2 × 103. 5 Less than 1.8 × 10 5 Less than 1.6 × 10 5 Less than 1.4 × 10 5 Less than 1.2 × 10 5 Less than one, 1×10 5 Less than 1, 9.5 × 10 4 Less than 1, 9×104 Less than 1, 8.5 × 10 4 Less than 1, 8×10 4 Less than 1, 7.5 × 10 4 Less than 1, 7×10 4 Less than 1, 6.5 × 10 4 Less than 1, 6×10 4 Less than 5.5 × 10 4 Less than 1, 5×10 4 Less than 1, 4.5 × 10 4 Less than 1, 4×10 4 Less than 1, 3.5 × 10 4 Less than 1, 3×10 4 Less than 1, 2.5 × 10 4 Less than 1, 2×10 4 Less than 1.5 × 10 4 Less than one, 1×10 4 Less than 1, 9.5 × 10 3 Less than 1, 9×10 3 Less than 1, 8.5 × 10 3 Less than 1, 8×10 3 Less than 1, 7.5 × 10 3 Less than 1, 7×10 3 Less than 1, 6.5 × 10 3 Less than 1, 6×10 3 Less than 5.5 × 10 3 Less than 1, 5×10 3 Less than 1, 4.5 × 10 3 Less than 1, 4×10 3 Less than 1, 3.5 × 10 3 Less than 1, 3×10 3 Less than 1, 2.5 × 10 3 Less than one, or 2 × 10 3 The number is below 1.

[0070] The volume of the aforementioned target area can range from 0.2 to 500 mm. 3 Preferably, it can be from 0.3 to 500 mm. 3 0.4 to 500 mm 3 The unit dosage is administered at predetermined intervals according to the area of ​​the target site. Furthermore, one of the aforementioned mesenchymal stem cell spheres may contain 200 to 800 mesenchymal stem cells, preferably 250 to 750 cells, more preferably 300 to 700 cells, and even more preferably 400 to 600 mesenchymal stem cells.

[0071] Specifically, based on the dosage per volume unit at the target site, 1×10-1 can be administered. 2 8 × 10 cells 7 The number of cells per cell, for example, the lower limit could be 1.0 × 10⁻⁶. 2 More than one cell, 1.2 × 10 2 More than one cell, 1.4 × 10 2 More than one cell, 1.6 × 10 2 More than one cell, 1.8 × 10 2 More than one cell, 2.0 × 10 2 More than one cell, 2.2 × 10 2 More than 10 cells, 2.4 × 10 2 More than one cell, 2.6 × 10 2 More than one cell, 2.8 × 10 2 More than one cell, 3.0 × 10 2 More than 10 cells, 3.2 × 10 2 More than one cell, 3.4 × 10 2 More than one cell, 3.6 × 10 2 More than one cell, 3.8 × 10 2 More than 10 cells, 4.0 × 10 2 More than 10 cells, 4.2 × 10 2 More than 10 cells, 4.4 × 10 2 More than one cell, 4.6 × 10 2 More than one cell, 4.8 × 10 2 More than one cell, 5.0 × 10 2 More than one cell, 6.0 × 10 2 More than one cell, 8.0 × 10 2 More than one cell, 1.0 × 10 3 More than one cell, 1.2 × 10 3 More than one cell, 1.4 × 10 3 More than one cell, 1.6 × 10 3 More than one cell, 1.8 × 10 3 More than one cell, 2.0 × 10 3 More than one cell, 2.2 × 10 3 More than 10 cells, 2.4 × 10 3 More than one cell, 2.6 × 10 3 More than one cell, 2.8 × 10 3 More than one cell, 3.0 × 10 3 More than 10 cells, 3.2 × 10 3 More than one cell, 3.4 × 10 3 More than one cell, 3.6 × 10 3More than one cell, or 3.8 × 10 3 More than one cell, with an upper limit of 8×10. 7 Less than 1 cell, 8.6 × 10 7 Less than 1 cell, 8.4 × 10 7 Less than 1 cell, 8.2 × 10 7 Less than one cell, 8×10 7 Less than 1 cell, 7.8 × 10 7 Less than 1 cell, 7.6 × 10 7 Less than 1 cell, 7.4 × 10 7 Less than 1 cell, 7.2 × 10 7 Less than one cell, 7×10 7 Less than 1 cell, 6.8 × 10 7 Less than 1 cell, 6.6 × 10 7 Less than 1 cell, 6.4 × 10 7 Less than 1 cell, 6.2 × 10 7 Less than one cell, 6×10 7 Less than 1 cell, 5.5 × 10 7 Less than one cell, 5×10 7 Less than 1 cell, 4.5 × 10 7 Less than one cell, 4×10 7 Less than 1 cell, 3.5 × 10 7 Less than one cell, 3×10 7 Less than 1 cell, 2.5 × 10 7 Less than one cell, 2×10 7 Less than 1 cell, 1.5 × 10 7 Less than one cell, 1×10 7 Less than one cell, 9×10 6 Less than one cell, 8×10 6 Less than one cell, 7×10 6 Less than one cell, 6×10 6 Less than one cell, 5×10 6 Less than one cell, 4×10 6 Less than one cell, 3×10 6 Less than 10 cells, 2.0 × 10 6 1 cell, or 1.5 × 10 6 Less than one cell.

[0072] The volume of the aforementioned target area can range from 0.2 to 500 mm. 3 Preferably, it can be from 0.3 to 500 mm. 3 0.4 to 500 mm 3 The dosage is administered at prescribed intervals based on the area of ​​the target site.

[0073] In this invention, after administration of the above-described composition, the target site is treated or restored, thereby enabling the treatment of skin regeneration, wounds, and scars. A single administration of the composition of this invention can exhibit skin regeneration, wound, and scar treatment effects for a considerable period after administration.

[0074] In this invention, skin regeneration can improve changes in the skin, such as deep or superficial wrinkles, caused by the passage of time, light, cosmetics, soap, pollution, dust, dryness, smoking, lifestyle habits, etc.

[0075] In this invention, the aforementioned scar can be any one or more selected from the following: scars caused by bedsores, scars caused by burns, scars caused by hair removal, scars caused by glandular scleroderma, scars caused by trauma, scars caused by diabetic foot ulcers, atrophic scars, and stretch marks. Furthermore, an example of the aforementioned scar can be a scar involving the epidermis, dermis, subcutaneous tissue, epidermis and dermis, dermis and subcutaneous tissue, or a scar involving damage to the epidermis, dermis, and subcutaneous tissue. Preferably, it can be a full-thickness scar that damages the dermis, or a full-thickness scar that damages the epidermis, dermis, and subcutaneous tissue.

[0076] In this invention, the term "wound" refers to bodily injury caused by a tissue cut, tear, break, burn, or traumatization, or by an obstacle or disease that induces such injury. The wound can be an open wound or a closed wound. An example of the wound is an open wound on the skin. An example of the wound is a wound involving damage to the epidermis; dermis; the epidermis and dermis; or the epidermis, dermis, and subcutaneous fat layer. Preferably, it is a full-thickness wound that damages the dermis or the epidermis, dermis, and subcutaneous fat layer.

[0077] Additionally, examples of the aforementioned wounds may include cuts, incisions (such as surgical incisions), trauma, abrasions, contusions, puncture wounds, fractures, lacerations, ruptures, burns, or amputations.

[0078] More specifically, the mesenchymal stem cell spheres according to the present invention can be administered directly to the scar site or wound site in a single dose, as described above.

[0079] The present invention provides a filler composition for skin regeneration comprising mesenchymal stem cell spheres.

[0080] This invention provides the use of mesenchymal stem cell spheres for the manufacture of a medicament for the treatment of skin wounds or scars, which can be applied topically to a target site or administered via subcutaneous or intradermal injection.

[0081] This invention provides mesenchymal stem cell spheres for local application or administration via subcutaneous or intradermal injection to target sites for the treatment of skin wounds or scars.

[0082] This invention provides a method for treating skin wounds or scars by topically applying or administering a therapeutically effective amount of mesenchymal stem cell spheres via subcutaneous or intradermal injection to a target site in a subject requiring treatment of skin wounds or scars.

[0083] The composition of the present invention, comprising mesenchymal stem cell spheres, can promote wound healing by direct administration to wounds, scars, or skin depressions in a therapeutically effective amount at sites requiring dermal or subcutaneous restoration and skin regeneration, and can also promote scar healing / regeneration and skin volume maintenance by inducing dermal and subcutaneous restoration within the skin. Attached Figure Description

[0084] Figure 1 This shows the morphology of mesenchymal stem cells isolated from adipose tissue as observed under a microscope.

[0085] Figure 2 This represents the cell growth rate of mesenchymal stem cells isolated from adipose tissue.

[0086] Figure 3 The purity of mesenchymal stem cells confirmed by flow cytometry is indicated, and negative and positive markers are shown.

[0087] Figure 4 This indicates the results of staining to verify the differentiation capacity of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

[0088] Figure 5 This indicates the results of RT-PCR verification of the differentiation capacity of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

[0089] Figure 6 The images shown are microscopic images of the culture of mesenchymal stem cells, which are round cell structures formed through self-assembly, i.e., mesenchymal stem cell spheres.

[0090] Figure 7 The size distribution of mesenchymal stem cell spheroids is represented by donor and passage number.

[0091] Figure 8 The cell viability of mesenchymal stem cell spheroids was indicated by donor and passage.

[0092] Figure 9 The amount of DNA in mesenchymal stem cell spheroids is expressed as a measure of culture time.

[0093] Figure 10 Electron microscope images of mesenchymal stem cell spheroids are shown.

[0094] Figure 11 The expression of mesenchymal stem cell markers in cells constituting mesenchymal stem cell spheroids was shown, as confirmed by flow cytometry.

[0095] Figure 12 The donor of the mesenchymal stem cell spheres is shown in a microscope image with immunofluorescence staining of mesenchymal stem cell markers.

[0096] Figure 13 The results show the results of analyzing the extracellular matrix expression levels in mesenchymal stem cell spheroids using quantitative RT-PCR.

[0097] Figure 14 A graph showing the expression of extracellular matrix in mesenchymal stem cell spheroids using enzyme-linked immunosorbent assay (ELISA) is displayed.

[0098] Figure 15 Images showing collagen expression in mesenchymal stem cell spheroids confirmed by fluorescent immunostaining are displayed.

[0099] Figure 16 Images showing collagen expression in mesenchymal stem cell spheroids, confirmed by histological analysis.

[0100] Figure 17 The results show the expression of growth factors in mesenchymal stem cell spheroids analyzed using real-time RT-PCR.

[0101] Figure 18 This is the result of collagen area analysis of the experimental and control groups of mesenchymal stem cell spheroids administered to a trauma-induced animal model. Figure 18 (A) is the result of intradermal injection of mesenchymal stem cell spheroids. Figure 18 (B) is the result of coating the drug-treated mesenchymal stem cell spheres.

[0102] Figure 19 The results show the combined scores of stability and efficacy indicators for the experimental and control groups of intradermal administration of mesenchymal stem cell spheres in a trauma-induced animal model.

[0103] Figure 20 The results of the wound area ratio analysis in the experimental and control groups of subcutaneously administered mesenchymal stem cell spheres in a trauma-induced animal model are shown.

[0104] Figure 21 The results of histopathological analysis of subcutaneously administered mesenchymal stem cell spheroids in a trauma-induced animal model are shown.

[0105] Figure 22 The results of area analysis of collagen and type I collagen in the experimental and control groups of normal nude mice treated with mesenchymal stem cell spheroids are shown.

[0106] Figure 23 The results of dermal thickness analysis in the experimental and control groups of normal nude mice treated with mesenchymal stem cell spheroids are shown.

[0107] Figure 24 The results show the results of in vitro experiments analyzing and comparing the ability of type 1 collagen and FN to generate in mesenchymal stem cell spheroids (TRTP-101), HDF, and MSC single cells (SC).

[0108] Figure 25 The results show the results of in vitro experiments comparing the protein expression levels of growth factors in mesenchymal stem cell spheroids (TRTP-101), HDF, and MSC single cells (SC).

[0109] Figure 26 The schematic diagram illustrates the application and restoration morphology of the mesenchymal stem cell spheres of the present invention on skin wounds or scars.

[0110] Figure 27 Antera 3D displays atrophic scar sites before and after administration of mesenchymal stem cell spheroids of the present invention (1 week, 4 weeks, and 8 weeks later). ® Take a picture.

[0111] Figure 28 The results are the volume measurements of atrophic scars before and after administration of the mesenchymal stem cell spheroids of the present invention (1 week, 4 weeks, and 8 weeks later).

[0112] Figure 29 This study demonstrates the inter-subject deviation in dry weight measurements of the mesenchymal stem cell spheres (MiB) of the present invention.

[0113] Figure 30 The linear trend line showing the dry weight of the mesenchymal stem cell spheres (MiB) of the present invention is displayed.

[0114] Figure 31 The results of the total protein count analysis of mesenchymal stem cell spheroids and single cells of the present invention are shown.

[0115] Figure 32 The results of the major component analysis of the mesenchymal stem cell spheroids and single cells of the present invention are shown.

[0116] Figure 33 The results of thermographic analysis of mesenchymal stem cell spheroids and single cells of the present invention are shown.

[0117] Figure 34 The diagram shows the distribution of protein increases and decreases in mesenchymal stem cell spheroids and single cells according to the present invention. Detailed Implementation

[0118] To achieve the above objectives, the present invention provides a method for manufacturing mesenchymal stem cells isolated from adipocytes into mesenchymal stem cell spheres.

[0119] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings and in a manner readily practiced by those skilled in the art. However, the present application can be implemented in various sizes and is not limited to the embodiments described herein.

[0120] The present invention will now be described in more detail through embodiments, but the embodiments described below are for illustrative purposes only and are not intended to limit the scope of the invention.

[0121] Example 1. Manufacturing and characterization of adipose-derived mesenchymal stem cell spheres (MiB)

[0122] Example 1-1. Isolation of mesenchymal stem cells (hMSCs) from adipose tissue

[0123] As the cell source for manufacturing the mesenchymal stem cell microblocks (MiB) of this invention, human adipose-derived mesenchymal stem cells (hMSCs), which are relatively easy to isolate and have a high growth rate, were used. The isolation of hMSCs was carried out after approval from the Seoul National University Dental Hospital Institutional Review Committee.

[0124] Prepared human adipose tissue was placed in Hanks® Balanced Salt Solution (HBSS) containing 0.1% Collagenase I. After separating the cells by shaking at 37°C for 30 minutes, the cells were washed and filtered through a 100 μm nylon mesh sieve. Nutrients were then provided using Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1X antibiotic antimycotic solution (AA), and the cells were cultured in a 5% CO2 incubator at 37°C.

[0125] Examples 1-2. Validation of isolated mesenchymal stem cells (hMSCs)

[0126] The hMSCs isolated in Example 1-1 have characteristics such as attachment morphology, multi-differentiation ability, and expression of surface-specific antigens. By confirming these characteristics, it is verified that the cells isolated in Example 1-1 are hMSCs.

[0127] The results confirming the morphological and distribution characteristics of the cells showed that the cells attached and grew in a homogeneous spindle-like shape in a plastic culture dish. Figure 1 And it proliferated exponentially until the 7th passage, confirming that each cell division takes about 2-3 days. Figure 2 ).

[0128] In addition, as a method to confirm whether the characteristics of hMSCs were well maintained through repeated passages, the expression of stem cell surface markers was confirmed by flow cytometry. Cultured MSCs were washed with PBS, treated with proteolytic enzymes such as trypsin and collagenase at 37°C for 5 minutes, and then recovered as single cells. Cells were then reacted with antibodies against fluorescently labeled CD14, CD29, CD31, CD34, CD44, CD73, CD90, HLA-DR, CD45, CD79α, CD105, and CD117 at 4°C for 1 hour. Then, after washing with Dulbecco's phosphate-buffered saline (DPBS) containing 2% FBS, the expression level of isotype mouse IgG relative to the control group was analyzed using FACS.

[0129] Analysis showed that over 95% of the cells were positive for the positive markers CD29, CD44, CD73, CD90, and CD105, while the expression of the negative markers CD31, CD45, CD79a, CD117, and HLA-DR was less than 2%, confirming that the cells were MSCs isolated at high purity. Figure 3 ).

[0130] Examples 1-3. Confirmation of the adipocyte differentiation capacity of isolated mesenchymal stem cells (hMSCs) by staining.

[0131] To confirm the adipocyte differentiation capacity, 9.5 x 10⁹ cells were used. 4 Cells were placed in 6-well plates using a high-glucose DMEM medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 100 nM dexamethasone, 100 μM indomethacin (INDO), 10 μg / mL insulin, 10% FBS, and 1X AA. The medium was changed twice a week, and the cells were cultured at 37°C in a 5% CO2 incubator for 14 days. To confirm differentiation, the culture medium from the differentiated plates was removed, and the cells were washed once with DPBS and fixed with 4% PFA at room temperature.

[0132] Then, to visually confirm differentiation, 0.2 g of oil red O powder was dissolved in 40 mL of isopropanol, and 1 mL of a solution prepared by filtering through a 0.22 μm filter was added dropwise. The reaction was carried out at room temperature for 15 minutes, and after washing five times with a DW (Dry Washing) solution, differentiation into adipogenic differentiation was confirmed. Figure 4 ).

[0133] Examples 1-4. Confirmation of osteocyte differentiation capacity of isolated mesenchymal stem cells (hMSCs) by staining.

[0134] To confirm the osteocyte differentiation capacity of hMSCs, 9.5 x 10⁻⁶ cells were subjected to the same method as in Examples 1-3 above. 4Cells were added to 6-well plates and cultured in a medium prepared by adding 10 nM dexamethasone, 50 μg / mL ascorbic acid, 10 mM β-glycerol phosphate, 1 mM dibutyryl-cAMP, 10% FBS, and 1X AA to alpha minimum essential medium (α MEM). After 4 days, the medium was completely removed, and the cells were cultured in medium without dibutyryl-cAMP, with the medium changed twice a week, in a 5% CO2 incubator at 37°C for 14 days, thereby inducing differentiation of hMSCs into osteocytes.

[0135] Then, to visually confirm osteoblast differentiation, alizarin red S staining was performed. After the culture medium in the plate where differentiation had ended, the cells were washed once with DPBS, and 1 mL of 4% paraformaldehyde (PFA) was added to fix the cells at room temperature. The fixative was removed, and the cells were washed with DPBS. 2 mL of a 1% alizarin red S solution diluted with distilled water (DW) was dispensed aliquoted and reacted at room temperature for 15 minutes. After washing five times with DW, the differentiation into osteoblasts was confirmed using an Olympus microscope with 1 mL of DW dispensed aliquots. Figure 4 ).

[0136] Examples 1-5. Confirmation of chondrocyte differentiation capacity of isolated mesenchymal stem cells (hMSCs) by staining.

[0137] To confirm chondrocyte differentiation capacity, 9.5 x 10⁹ cells were cultured using the same method as in Examples 1-3 above. 4Cells were placed in 6-well plates and cultured in a medium containing 1X insulin transferrinselenium premix (ITS), 50 ng / mL ascorbic acid, 40 μg / mL L-proline, 100 nM dexamethasone, 10% FBS, and 1X AA in α-MEM, with 10 ng / mL transforming growth factor (TGF-β3) added. The plates were incubated at 37°C in a 5% CO2 incubator for 21 days. After differentiation, the culture medium was removed from the plates, and the cells were fixed at room temperature using 4% PFA.

[0138] After cell fixation, the cells were washed with DPBS and placed in an OCT compound, then frozen at -80°C for one day. The frozen cells were then sectioned into 8 μm thick sections using a cryotome and washed with dry whey (DW). A 1% alcian blue solution (prepared using alcian blue reagent) was added to the sections, and staining was performed at room temperature for 30 minutes. The sections were then washed with 0.1N HCl solution and washed with dry whey (DW) to confirm differentiation into chondrogenic cells. Figure 4 ).

[0139] Examples 1-6. RT-PCR was used to verify the differentiation capacity of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

[0140] Total RNA was isolated from cells using chloroform and TRI reagent. cDNA was synthesized from 1 μg of total RNA using Prime Script RTMaster Mix. The synthesized cDNA was subjected to PCR using an AccuPower PCR PreMix (Bioneer) PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were analyzed by agarose gel electrophoresis. Figure 5 The primer sets used are shown in Table 1.

[0141] [Table 1]

[0142]

[0143] The results of the differentiation capacity confirmed by the above Examples 1-3 to 1-6 confirm that the cells isolated from human adipose tissue by the method of the present invention have very high purity and retain the characteristics of stem cells even after long-term culture.

[0144] Examples 1-7. Manufacturing of mesenchymal stem cell spheres (MiB) via self-assembly of mesenchymal stem cells (MSCs)

[0145] To facilitate MSC self-assembly, MSCs cultured in a culture vessel were washed with PBS, treated with proteolytic enzymes such as trypsin and collagenase, and recovered as single cells. Cells were seeded in wells, centrifuged, and cultured under physical pressure in a 3D culture vessel at 37°C for 1, 2, 3, and 7 days. Over time, round cell structures of a certain size, namely mesenchymal stem cell spheroids (also named MiB in this specification), were generated. Figure 6 Mesenchymal stem cell (MSC) spheroids were passed through double-layer cell strainers with pore sizes of 70 μm and 300 μm to collect uniformly sized MSC spheroids. The MSC spheroids were imaged using the JuLi stage system, and the MiB diameter was measured using ImageJ software. No significant difference in MSC spheroid diameter was observed between donor and passage number. Each well produced MSC spheroids ranging in diameter from a minimum of 40 μm to a maximum of 260 μm, with an average diameter of 98.74 μm–116.81 μm. Figure 7 ).

[0146] Examples 1-8. Cell viability of mesenchymal stem cell spheroids (MiB) under donor and passage differences.

[0147] To investigate the viable cells within mesenchymal stem cell spheroids, cell viability was determined using the LIVE / DEAD™ Viability / Cytotoxicity Kit and the MTT Cell Proliferation Assay Kit. Using the LIVE / DEAD Viability / Cytotoxicity Kit, viable cells were stained green, and dead cells were stained red. The results showed that most cells constituting the mesenchymal stem cell spheroids were viable, while cells not well-bound or weakly bound to the outside of the spheroids were stained red. Figure 8 Compared to two-dimensional culture, live cells using MSCs passaged 3 times showed an 84% viability, and cell viability did not change significantly between passages. Figure 8 This indicates that cells existing in a three-dimensional environment survive more stably.

[0148] Examples 1-9. Confirmation of cell growth by DNA content analysis of mesenchymal stem cell spheroids (MiBs).

[0149] The DNA content of mesenchymal stem cell spheroids was determined using the Quant-iT™ PicoGreen™ dsDNA assay kit. The DNA content of the mesenchymal stem cell spheroids did not change significantly during the culture period from day 1 to day 7. Figure 9 ).

[0150] These results indicate that no significant cell proliferation was detected during the culture of mesenchymal stem cell spheroids.

[0151] Examples 1-10. Mesenchymal stem cell spheroids observed using scanning electron microscopy (SEM).

[0152] After fixing the mesenchymal stem cell spheroids, they were treated with HCl-EtOH solution. The treated mesenchymal stem cell spheroid samples were then fixed with 1% osmium tetroxide, dehydrated, and chemically dried using hexamethyldisilazane (HMDS). They were then coated with a platinum layer using a Q150TS microscope. SEM images were obtained using an ApreoS spectrometer at 10 kV. SEM observation of the mesenchymal stem cell spheroids revealed a very dense, spherical structure with a very rough surface and numerous vesicles forming on the cell surface. Figure 10 ).

[0153] Examples 1-11. Confirmation of the mesenchymal stem cell characteristics of the cells constituting the prepared mesenchymal stem cell spheroids

[0154] To confirm whether the cells constituting mesenchymal stem cell spheroids (MiBs) still maintain MSC characteristics, the MiBs were washed with PBS and treated with proteolytic enzymes such as trypsin and collagenase to separate them into single cells. The expression of surface proteins of the separated cells was studied using the aforementioned flow cytometry method.

[0155] The study results showed that the cells constituting mesenchymal stem cell spheroids expressed more than 95% of the positive surface markers of MSCs, including CD29, CD44, CD73, CD90, and CD105, while the expression of negative surface markers CD31, CD45, CD79a, CD117, and HLA-DR was less than 2%. Figure 11 ).

[0156] In addition, mesenchymal stem cell spheroids (MiBs) were fixed using the Image-iT Fixation / Permeabilization Kit to prevent degradation and improve cell membrane permeability. Afterward, they were cultured for 18 hours at room temperature (RT) with 3% bovine serum albumin (BSA) in PBS and antibodies against MSC markers (CD73, CD90, CD105). After washing with PBS, the MiBs were cultured with fluorescently labeled secondary antibodies at room temperature for 6 hours. The nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature. This confirmed that most cells in the fluorescently stained MiBs expressed the MSC markers CD73 (red), CD90 (green), and CD105 (green). Figure 12 ).

[0157] Examples 1-12. Analysis of extracellular matrix expression levels in mesenchymal stem cell spheroids by quantitative RT-PCR

[0158] When MSCs morphologically transform into mesenchymal stem cell spheroids after three-dimensional culture, the changes in extracellular matrix gene expression were investigated using quantitative real-time RT-PCR. As in Examples 1-6, RNA was isolated from mesenchymal stem cells (SCs) or mesenchymal stem cell spheroids (MiBs) cultured from day 1 to day 7 using TRI reagent, and cDNA was synthesized using Prime Script RT Master Mix. PCR reactions were performed using 2X qPCRBIO SyGreen MiX Hi-ROX. Primers for COL1A1 (collagen type I), COL3A1 (collagen type 3), ELN (elastin), and FN1 (fibronectin) were prepared using QuantiTect primer Assays (Qiagen), with GAPDH used for expression level correction. The results are as follows: Figure 13 As shown.

[0159] from Figure 13It was confirmed that fibronectin expression increased rapidly with prolonged culture time of mesenchymal stem cell spheroids, and type I collagen increased on day 1 of culture compared to 2D cultured mesenchymal stem cells. Furthermore, the expression of ECM-related genes was increased in the mesenchymal stem cell spheroids of this invention, and it is expected that ECM production may occur from MSCs within the mesenchymal stem cell spheroids even after at least 7 days of drug administration.

[0160] Examples 1-13. Analysis of extracellular matrix expression levels in mesenchymal stem cell spheroids by enzyme-linked immunosorbent assay (ELISA).

[0161] The mesenchymal stem cells or mesenchymal stem cell spheroids of the present invention were decomposed using a mammalian protein extractio reagent containing 1 mM phenylmethanesulfonyl fluoride and a protein inhibitor cocktail. After centrifugation at 16000 x g for 20 minutes at 4°C, type I collagen, fibronectin, and hyaluronic acid were analyzed using an ELISA (enzyme-linked immunosorbent assay) kit. Type I collagen, fibronectin, and hyaluronic acid accumulated in the mesenchymal stem cell spheroids (MiB). Figure 14 ).

[0162] Examples 1-14. Confirmation of collagen expression in mesenchymal stem cell spheroids by fluorescent immunostaining.

[0163] To confirm collagen expression in the mesenchymal stem cell spheroids (MiBs) of this invention, paraffin-embedded MiBs were cut into 4 μm thick sections and slides were prepared. After dewaxing and hydration of the slides, the MiBs were immunostained with a fluorescently labeled type 1 collagen antibody to analyze collagen expression. The results are as follows: Figure 15 As shown.

[0164] from Figure 15 It can be confirmed that, compared with single cells, the expression of type I collagen is significantly higher in mesenchymal stem cell spheroids (MiB). Figure 15 ).

[0165] Examples 1-15. Histological analysis confirmed collagen expression in mesenchymal stem cell spheroids.

[0166] To confirm collagen expression in the mesenchymal stem cell spheroids (MiB) of the present invention via histological analysis, as in Examples 1-14, paraffin-embedded mesenchymal stem cell spheroids were cut into 4 μm thicknesses to prepare slides. After deparaffining and hydration of the slides, immunohistochemistry (IHC) staining was performed using a primary antibody against type 1 collagen and a biotinylated secondary antibody. This confirmed that collagen in the mesenchymal stem cell spheroids was stained brown. Figure 16 A).

[0167] In addition, the use of a trichrome staining kit confirmed the presence of collagen in the mesenchymal stem cell spheroids by identifying the areas stained blue. Figure 16 B).

[0168] Examples 1-16. Analysis of growth factor expression in mesenchymal stem cell spheroids by quantitative RT-PCR

[0169] The expression of growth factors in the mesenchymal stem cell spheroids (MiB) of the present invention was analyzed. Specifically, to investigate the gene expression of fibroblast growth factor 2 (FGF2), platelet-derived growth factor subunit A (PDGFA), vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1) in the mesenchymal stem cell spheroids (MiB), quantitative RT-PCR was performed as described in Examples 1-12. Primers were designed as shown in Table 2, and the quantitative RT-PCR mentioned above was performed. The results are as follows: Figure 17 As shown.

[0170] [Table 2]

[0171]

[0172] The analysis results showed that the expression of multiple growth factors had a positive effect on skin regeneration. For example... Figure 17 As shown, the expression levels of FGF2 and PDGFA in mesenchymal stem cell spheres (MiB) were reduced, while the expression levels of VEGFA and HGF were increased, indicating that mesenchymal stem cell spheres contribute to angiogenesis and regeneration of damaged tissues.

[0173] Example 2. Efficacy test using an animal model

[0174] Example 2-1. Confirmation of the therapeutic effects of skin regeneration, skin wounds, or scars using a trauma-induced animal model.

[0175] (1) Evaluation of the collagen production capacity of Masson's Trichrome (MT)

[0176] A full-thickness wound-induced trauma model was created using a scalpel. The subcutaneous tissue was bluntly dissected, symmetrically around the rat's spinal line, to avoid damage to the muscle layer, until the dermis was removed. The wound-induced rats, as shown in Table 3, were treated with intradermal, subcutaneous, and topical administration of low (0.5 x 10⁻⁶) drugs. 6 (cells), in (1.0x10) 6 (number of cells), height (2.0 x 10⁻⁶) 6 (per cell) dose of TRTP-101 and TRTP-101 excipients. Then, after applying 3M's Tegaderm to protect the wound site, collagen area, etc., were checked on day 21.

[0177] After staining skin tissue samples with Masson's Trichrome (MT), the collagen area in the blue region was measured using the ZEN analysis software on three randomly selected images from the corresponding tissue. The results are expressed as a percentage of the collagen-stained area relative to the total area of ​​the image. Figure 18 As shown. Figure 18 (A) indicates the result of intradermal administration. Figure 18 (B) indicates the result when the drug is applied.

[0178] [Table 3]

[0179]

[0180] from Figure 18 (A) It can be confirmed that, in terms of collagen area, the adipose-derived mesenchymal stem cell spheroids administration group of the present invention significantly increased to 51% in the low-dose group and 50% in the medium-dose group, compared to 44% in the excipient-administered group. This confirms that, in the experimental group given the adipose-derived mesenchymal stem cell spheroids of the present invention on day 21 of the remodeling step, collagen production was statistically significantly increased.

[0181] In addition, from Figure 18(B) It can be confirmed that, when administered via a coating method, in terms of collagen area, the adipose-derived mesenchymal stem cell spheroids administration group of the present invention showed a significant increase to 53% in the low-dose group, a significant increase to 50% in the high-dose group, and a trend towards an increase to 47% in the medium-dose group, compared to 40% in the excipient-administered group. Therefore, it is shown that the experimental group given the adipose-derived mesenchymal stem cell spheroids of the present invention generated more collagen compared to the control group.

[0182] Table 4 below summarizes the results of intradermal or topical administration of the adipose-derived mesenchymal stem cell spheroids of the present invention to normal rat skin collagen levels and to trauma-induced rats. Table 4 confirms that, when comparing normal collagen levels in rats with those under TRTP-101 administration (unit: %), compared to approximately 62% collagen content in the skin of 10-week-old normal rats (Song et al.), the adipose-derived mesenchymal stem cell spheroids of the present invention showed significant tissue remodeling with 50% collagen recovery and up to 81% on day 21 after trauma.

[0183] [Table 4]

[0184]

[0185] (2) Evaluation of skin regeneration and wound healing during intradermal and subcutaneous administration

[0186] To evaluate skin regeneration and wound healing in intradermal drug administration trials, four indicators—inflammation level, necrosis, epithelialization, and collagenization—were evaluated in hematoxylin and eosin (H&E) stained slides, and two indicators—collagen fiber density and collagen fiber arrangement—were evaluated in MT-stained slides, for a total of six indicators. A total score was calculated for each rat on a scale of 0-5, with lower scores assigned as skin regeneration and wound healing improved, to assess significance. The results of the combined scoring of skin regeneration and wound healing indicators are shown in Table 5 below. Figure 19 As shown. Refer to Table 5 and Figure 19 The results showed that when administered intradermally, the medium-dose group exhibited significantly greater skin regeneration and wound healing compared to the excipient-administered group.

[0187] [Table 5]

[0188]

[0189] Each time point is represented as the mean + SE (n=10).

[0190] p<0.05, indicating a significant difference compared to the normal control group (G1) as detected by Student's t-test.

[0191] In addition, the results of analyzing the wound area ratio to evaluate the degree of skin regeneration and wound healing in the subcutaneous administration trial were used to analyze TRTP-101 1.0x10 6 The ratio of wound area on days 2 to 4 after administration in the cell / head dosing group (G2) showed a decreasing trend compared to the induction control group (G1). In particular, the wound area on day 3 was confirmed to be significantly smaller compared to the excipient dosing group. Figure 20 Based on the above results, it is believed that administration of TRTP-101 to the wound site can aid in the early stages of wound recovery.

[0192] In addition, histopathological examination results, which evaluate and score four indicators—inflammation level, necrosis, epithelialization, and collagenization—are as follows: Figure 21 As shown. From Figure 21 It can be confirmed that the levels of inflammation and necrosis are reduced, and the promotion of epithelialization and collagen production in the subcutaneous TRTP-101 administration group (G2) was significantly increased compared with the excipient administration group.

[0193] Example 2-2. Efficacy test using normal nude mice.

[0194] (1) Analyze the collagen positive area and type 1 collagen positive area based on the dosage.

[0195] On the left / right sides of the back of BALB / c nude mice that only lacked mature T cells and whose immune function was not completely suppressed, low (0.25x10) 6 (G2), (0.5x10) 6 (G3), High (1.0x10) 6 TRTP-101 (G4) and the excipient (G1) were each administered intradermally at 0.1 mL / site to two sites to evaluate in vivo persistence. The results are as follows: Figure 22 and Figure 23 As shown.

[0196] from Figure 22 It can be confirmed that, 4 weeks after administration, compared with the control group, the synthesis of collagen and type I collagen, which play a structural supporting role, showed a significant increase in the experimental group given the adipose-derived mesenchymal stem cell spheres of the present invention (each point is expressed as mean + SE (n=3~6)). p<0.05, p<0.01, indicating a significant difference compared to the excipient-treated group as detected by Student's t-test.

[0197] (2) Continuous in vivo evaluation

[0198] like Figure 23 As shown, at week 8, the dermal thickness of the mice was significantly increased compared to the control group. Furthermore, the dermis of the medium-dose group became the thickest, which was also significantly different from the control group.

[0199] Example 3. Efficacy test through in vitro experiments

[0200] Example 3-1. Confirmation of ECM generation capability

[0201] To confirm the extent of ECM-related substance production as an in vitro efficacy indicator, the production capacity of Type I collagen and FN from the adipose-derived mesenchymal stem cell spheroids (TRTP-101) of this invention was compared with that of the same number of HDF (human dermal fibroblasts) and MSC single cells (SCs) using ELISA analysis. The quantification results are as follows: Figure 24 As shown.

[0202] like Figure 24 As shown, the amounts of Type I collagen and FN were significantly increased in the mesenchymal stem cell spheroids of the present invention compared to HDF (human dermal fibroblasts) and SC. The expression levels of Type I collagen and fibronectin, which forms the supporting network by combining components of ECM, were approximately 7-fold and 11-fold higher, respectively, compared to HDF. The adipose-derived mesenchymal stem cell spheroids of the present invention generated more ECM in vitro compared to HDF (human dermal fibroblasts).

[0203] As a result, when the fat-derived mesenchymal stem cell spheres of the present invention are applied in vivo and in the human body, they generate type I collagen, the largest component of dermal tissue, to support the skin and connective tissue. Through cell attachment, growth, and wound healing, the FN is expected to have a volume-building effect on atrophic scars through treatment and regeneration.

[0204] Example 3-2. Confirmation of growth factor secretion capacity

[0205] The gene expression levels of VEGFA and HGF were analyzed using qRT-PCR, confirming that, in vitro, the adipose-derived mesenchymal stem cell spheroids of this invention exhibited higher expression of growth factors compared to HDF (human dermal fibroblasts) and MSC single cells (SCs). The results are as follows: Figure 25 As shown.

[0206] like Figure 25 As shown, the protein expression levels of VEGF and HGF in the adipose-derived mesenchymal stem cell spheroids of the present invention are approximately 9.5-fold and 75-fold higher, respectively. Therefore, it was confirmed that when the adipose-derived mesenchymal stem cell spheroids of the present invention were applied in an in vivo wound model, the rapid recovery observed on day 3, the initial stage of wound healing, was due to the high expression of wound-healing-related growth factors in the adipose-derived mesenchymal stem cell spheroids of the present invention.

[0207] Example 4. Drug resistance, stability, and efficacy trial in patients with atrophic scars.

[0208] The efficacy of TRTP-101 (adipose-derived mesenchymal stem cell spheroids) was confirmed in patients aged 19 years and older with atrophic scars who wished to undergo scar repair, following a single dose. The study included patients with four or more scars measuring 0.4 mm or more. 3 Patients with atrophic scars of the above volume were treated. Subjects may include patients who can be classified using the KCD disease code L90.

[0209] TRTP-101 (fat-derived mesenchymal stem cell spheres) was prepared using fat collected from the abdomen or thigh of the subjects. After a single dose to the subjects, stability, drug resistance, and efficacy were confirmed within a specified time.

[0210] After local anesthesia with 1% lidocaine in the patient's abdomen or thigh, 60-100 mL of tumescent solution (0.9% normal saline, 2% lidocaine, 0.1% epinephrine, and 8.4% sodium bicarbonate) was injected through a cannula for liposuction, collecting more than 60 mL of fat. Autologous adipose-derived mesenchymal stem cells were then selected from this fat and proliferated extensively. TRTP-101 (adipose-derived mesenchymal stem cell spheroids) were then created through three-dimensional culture, maintaining a size of 0.5-4.0 × 10⁻⁶. 7 Cells / mL.

[0211] After suspending the cells in the syringe by gently agitating them on a culture medium, sterilize the injection site and administer a single intradermal injection using a 29 or 30 gauge needle. Administer the medication in a way that ensures tautness at the atrophic scar site, and administer at least 0.4 x 10 at each site. 3 Cells, up to 2×10 6 Each cell.

[0212] Exploratory efficacy was analyzed for subjects in clinical trials of pharmaceuticals for whom data on exploratory efficacy evaluation variables were available. Antera 3D was used. ® Photos of the scar sites were taken before and after administration of TRTP-101 (adipose-derived mesenchymal stem cell spheres). Figure 27 The volume of depression was measured, and the results showed a reduction in the location of depression scars. Figure 28 To document the administration sites of the clinical trial pharmaceuticals, photographs showing the administration sites were taken and stored along with supporting documentation. The rate of change in the administration sites before and after administration was determined by measuring the volume of atrophic scars at each time point.

[0213] Reference Figure 28 Eight weeks after administration, the size of the scars showed a significant reduction compared to before administration.

[0214] To confirm drug resistance and stability, no adverse events occurred during the 4-week period following TRTP-101 administration. The adverse events were classified as Grade 3 or higher according to the Common Terminology Criteria for Adverse Events (CTCAE) Ver. 5.0, corresponding to the dose-limiting toxicity (DLT). All adverse events were analyzed by decomposition and classification according to the latest version of MedDRA's System Organ Class (SOC) and Preferred Term (PT).

[0215] The clinical trial results of Example 4, as shown in the efficacy trials utilizing animal models, demonstrated a significant or marked effect on the treatment of atrophic scars. Specifically, in the TRTP-101 administration group of the pharmaceutical composition of the present invention comprising adipose-derived mesenchymal stem cell spheroids, the volume of atrophic scars was reduced to a degree that showed a significant therapeutic effect.

[0216] Example 5. Confirmation of the physical properties of the mesenchymal stem cell spheres (MiB) of the present invention

[0217] (1) Wet weight analysis

[0218] To confirm the in vitro properties of the mesenchymal stem cell spheroids manufactured according to Example 1 (referred to as MiB or TRTP-101 in this invention), a wet weight analysis was performed. Specifically, to confirm the weight of the mesenchymal stem cell spheroids according to the invention, the total weight of 1200 spheroids produced was measured to estimate the weight of each spheroid, and the results are shown in Table 6 below.

[0219] [Table 6]

[0220]

[0221] The test results confirmed that the wet weight of each mesenchymal stem cell sphere according to the present invention was approximately 3.19 ± 0.46 μg.

[0222] (2) Dry weight analysis

[0223] To determine the accurate weight of the mesenchymal stem cell spheres according to the present invention, excluding the influence of solvents such as culture medium or buffer solutions, an experiment was conducted to determine the dry weight using a freeze-drying method.

[0224] ① Workflow for dry weight determination

[0225] To confirm one of the characteristics of in vitro mesenchymal stem cell spheroids (MiB), namely dry weight, the experimental workflow is as follows. For the mesenchymal stem cell spheroids (MiB) according to the present invention, two experimenters conducted the experiments separately to confirm inter-experimental bias. Cells were recovered at 70% confluence, and 0.5K mesenchymal stem cell spheroids (MiB) were nutrient-fed with DMEM containing 10% fetal bovine serum and 1X antibiotic antifungal solution and cultured for 2 days in a 5% CO2 culture vessel at 37°C. They were grouped as shown in Table 7 below.

[0226] [Table 7]

[0227]

[0228] During the culture of mesenchymal stem cell spheroids (MiB), the 5 mL tubes and parafilms used for recycling were weighed using an electronic scale before recycling. Each group was weighed three times using the electronic scale, and the average of the three measurements was calculated to determine the final weight. Before each measurement, the electronic scale was zeroed before weighing; if a weight difference of more than 0.2 μg was confirmed compared to the previous measurement, the scale was zeroed again before weighing.

[0229] Mesenchymal stem cell spheroids (MiBs) cultured for 48 hours at the preparation time were collected in 5 mL tubes (0, 4800, and 9600 samples, respectively) and suspended in 200 μL of DPBS. The openings of the 5 mL tubes were sealed with parafilm, punctured with a 30-gauge needle, and then freeze-dried for 2 days. The freeze-dried samples were weighed three times using an electronic scale, and the average of the measurements was calculated to determine the total weight. The weight of the 5 mL tubes and parafilm was subtracted from the weight of the freeze-dried samples to determine the dry weight of 4800 and 9600 MiBs, respectively. This calculated dry weight was then divided by the number of MiBs to determine the weight of one MiB.

[0230] ② Results of dry weight determination of mesenchymal stem cell spheroids (MiB) and singlets of mesenchymal stem cell spheroids (MiB).

[0231] The dry weight measurements of 4800 / 9600 mesenchymal stem cell spheroids (MiBs) and the dry weight measurements of individual MiBs, determined according to the dry weight measurement workflow, are shown in Table 8 below. The deviation in the dry weight measurement of a single MiB between the two experimenters is as follows: Figure 29 As shown, the linear trend results of mesenchymal stem cell spheroid (MiB) weight are as follows: Figure 30 As shown.

[0232] [Table 8]

[0233]

[0234] Refer to Table 8 above and Figure 29 The weights of the mesenchymal stem cell spheres (MiB) of the present invention measured by Experimenter 1 and Experimenter 2 were 336.2±30.5ng and 322.9.2±17.6ng (mean ± SD), respectively, with no statistically significant difference.

[0235] In addition, the dry weight data of 0, 4800, and 9600 mesenchymal stem cell spheroids from the two subjects were integrated, and the linear trend line was calculated using R0. 2 =0.9917 indicates that the experiment is statistically significant. Figure 30 ).

[0236] Additionally, the dry weight of one mesenchymal stem cell sphere (MiB), calculated using the trend line, is 317 ng, which is shown to be within the range of mesenchymal stem cell spheres (MiB) measured in Table 8.

[0237] Example 6. Proteomic analysis of mesenchymal stem cell spheroids (MiB) of the present invention

[0238] (1) Analysis of the number of proteins constituting single cells and mesenchymal stem cell spheroids according to the present invention

[0239] To confirm the characteristics of the mesenchymal stem cell spheres according to the present invention compared to single cells, proteomic analysis was performed on the fabricated mesenchymal stem cell spheres and single cells.

[0240] For this analysis, following the method of Lee, Jangho, et al., the sample was placed in a lysis buffer containing 4% SDS, boiled at 100°C for 10 minutes, and then centrifuged at 18,000 rpm for 10 minutes to extract the protein. The protein was then determined using the Bicinchoninica acid (BCA) assay, separated by SDS-PAGE gel, and subjected to in-gel digestion. Following the method of Lee, Sang-Yeop, et al., the gels were fractionated according to molecular weight and washed with a solution prepared from 30% methanol, 10 mM ammonium bicarbonate, and 50% acetonitrile. After washing, the gel was dried, reduced with 10 mM dithiothreitol and 55 mM iodoacetamide, and then subjected to tryptic digestion with 50 mM ammonium bicarbonate at 37°C for 12–16 hours. Tryptic peptides were extracted using a 50 mM ammonium bicarbonate solution containing 5% trifluoroacetylacid (TFA) and 50% acetonitrile, and stored at 4°C.

[0241] The solution preserved according to the above method was dissolved in 0.5% TFA and analyzed by liquid chromatography-tandem mass spectrometry (LC–MS / MS). 5 μL of the dissolved sample was dissolved for 95 minutes in 5%–40% acetonitrile using a 100 μm × 2 cm nanopiper trap column and a 75 μm × 15 cm nanopiper analysis column (Thermo Fisher Scientific) at a flow rate of 300 nL / min. All MS and MS / MS spectral data were analyzed using a QExactive Plus mass spectrometer (Thermo Fisher Scientific).

[0242] Protein identification was based on monoisotopic mass selection, a precursor mass tolerance of ±5 Da, a fragment mass tolerance of ±0.8 Da, and confirmation using two instances of incorrect cleavage and modification of carbamidomethylcysteine. Individual spectra were searched through various databases, specifically included based on a calculated Mascot ion threshold fraction of 0.05. A peptide validator was used to match peptides and proteins exceeding the threshold, with a false discovery rate (FDR) of 1.0%.

[0243] Single cells and the mesenchymal stem cell spheroids (MiB) of this invention were tested with three samples per group, and the results are as follows: Figure 31 As shown.

[0244] from Figure 31 It can be confirmed that the analysis of the number of proteins constituting single cells and the mesenchymal stem cell spheres (MiB) of the present invention confirms that the total number of proteins constituting the mesenchymal stem cell spheres (MiB) and single cells increases slightly by about 3%.

[0245] (2) Analysis of the main components of single cells and mesenchymal stem cell spheroids according to the present invention

[0246] To identify intergroup relationships and differences in protein composition using the proteins that make up the samples, principal component analysis (PCA) and heatmap analysis were performed. The PCA distribution chart showing the relationships between constituent proteins confirmed that single-cell and mesenchymal stem cell spheroids were located in opposite directions, indicating significantly different types of constituent proteins. The results are as follows: Figure 32 As shown. Furthermore, similarly, heatmap analysis, which used rows, columns, and colors to represent the relationships between groups, confirmed differences in protein composition and content between single cells and mesenchymal stem cell spheroids, as shown in the results. Figure 33 As shown.

[0247] (3) Analysis of the distribution of protein increase and decrease in mesenchymal stem cell spheroids according to the present invention

[0248] The distribution of protein increases and decreases in the mesenchymal stem cell spheroids (MiB) of the present invention relative to single cells was analyzed. The distribution of protein increases and decreases is illustrated in the figure below. Figure 34The results confirming the increase in structural proteins in the mesenchymal stem cell spheres (MiB) of the present invention relative to single cells are shown in Table 9.

[0249] [Table 9]

[0250]

[0251] The analysis results of the volcano plot, which visually demonstrates the statistical significance of changes in protein composition, are as follows ( Figure 34 Compared to single cells, the most significant change observed in the mesenchymal stem cell spheroids (MiB) of this invention is in tenascin (TNC). Tenascin is a protein expressed when tissue is damaged or inflamed, and it is known to participate in cell proliferation responses and promote healing by activating the epidermal growth factor receptor (EGFR) and inflammatory signaling pathways.

[0252] Similarly, fibronectin (FN1) is one of the proteins that has been identified as significantly increased. Fibronectin is known to play an important role in wound healing and tissue remodeling by participating in cell attachment, growth, migration and differentiation.

[0253] As described above, the proteins added to the mesenchymal stem cell spheroids (MiB) of the present invention relative to single cells are functionally classified into structural proteins, enzymes, ribosomal proteins, histones, signal transduction proteins, apoptosis and differentiation-related proteins, etc. In particular, the increase in structure-related proteins is presumed to be due to changes caused by the self-assembly induction principle of the generation of the mesenchymal stem cell spheroids (MiB) of the present invention.

[0254] Furthermore, proteoglycan-4 (PRG4) has been shown to be a protein specifically expressed only in mesenchymal stem cell spheroids (MiBs). PRG4 is known to induce adipocytes to the wound site by regulating molecular pathways related to wound healing, thereby aiding wound healing through reduced fibrosis and enhanced angiogenesis.

[0255] Furthermore, proteins that provide structural support in tissues such as skin, including COL5A3 (Collagen Type V Alpha 3 Chain), COL7A1 (Collagen Type VII Alpha 1 Chain), EVPL (Envoplakin), EFEMP2 (Fibulin-4), and MFAP2 (Microfibril Associated Protein 2), were confirmed to be newly generated in mesenchymal stem cell spheroids (MiBs) compared to single cells.

[0256] This protein analysis method confirmed that a variety of structural proteins were specifically expressed in the mesenchymal stem cell spheres (MiB) of the present invention, and that extracellular matrix proteins were also increased.

Claims

1. A pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheres, said mesenchymal stem cell spheres being topically applied to a target site or administered via subcutaneous or intradermal injection.

2. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheres are three-dimensional cell aggregates of mesenchymal stem cells.

3. The pharmaceutical composition according to claim 1, wherein, The diameter of the mesenchymal stem cell spheres is 10 to 800 μm.

4. The pharmaceutical composition according to claim 1, wherein, The wet weight of the mesenchymal stem cell spheroids is 1 to 6 μg.

5. The pharmaceutical composition according to claim 1, wherein, The dry weight of the mesenchymal stem cell spheroids is 250 to 500 ng.

6. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cells are selected from any one of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and peripheral blood-derived mesenchymal stem cells.

7. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheres comprise mesenchymal stem cells, i.e., MSCs, and extracellular matrix, i.e., ECM.

8. The pharmaceutical composition according to claim 7, wherein, The extracellular matrix contains one or more of the following: collagen, fibronectin, hyaluronic acid (HA), elastin (EL), and glycosaminoglycans (GAG).

9. The pharmaceutical composition according to claim 8, wherein, The collagen is type 1 collagen.

10. The pharmaceutical composition according to claim 1, wherein, In the mesenchymal stem cell spheroids, the expression of any one or more of the following growth factors is increased: fibroblast growth factor (FGF2), platelet-derived growth factor (PDGFA), vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1).

11. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheroids comprise cells that positively express at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any one or more markers selected from CD29, CD44, CD73, CD90, and CD105.

12. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheroids comprise cells that negatively express at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2% or less of any one or more markers selected from CD31, CD45, CD79a, CD117 and HLA-DR.

13. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheroids comprise cells that express at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% positive expression of CD29, CD44, CD73, CD90, and CD105, and at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, or 2% negative expression of CD31, CD45, CD79a, CD117, and HLA-DR, or less.

14. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheroids express one or more proteins selected from tendinin (TNC), fibronectin (FN1), proteoglycan (PRG4), COL5A3 (type V collagen α-3 chain), COL7A1 (type VII collagen α-1 chain), EVPL (pigmentin), EFEMP2 (fibrinogen-4), MFAP2 (microfibril-associated protein 2), FBLN2 (fibrinogen-2), HSPG2, FBLN1 (fibrinogen-1), and COL6A3 (type VI collagen α-3 chain).

15. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition is administered once, twice, or three times.

16. The pharmaceutical composition according to claim 1, wherein, The administration involves directly delivering the drug to the target area where skin regeneration through collagen production is needed.

17. The pharmaceutical composition according to claim 1, wherein, The target area includes parts of the skin where the dermis is damaged or depressed.

18. The pharmaceutical composition according to claim 1, wherein, The target area includes the subcutaneous layer of the skin that is damaged or depressed.

19. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheres induce dermal regeneration within the skin, thereby promoting skin regeneration.

20. The pharmaceutical composition according to claim 1, wherein, The mesenchymal stem cell spheres induce subendothelial restoration in the skin, thereby promoting skin regeneration.

21. The pharmaceutical composition according to claim 1, wherein, The dosage form of the pharmaceutical composition is selected from any one of the following: injection, infusion, spray, liquid, suspension, external application, or patch.

22. The pharmaceutical composition according to claim 1, wherein, The unit dosage contains 1 to 2 × 10 5 The mesenchymal stem cell spheres described above.

23. The pharmaceutical composition according to claim 1, wherein, The scar is selected from any one or more of the following: scars caused by bedsores, scars caused by burns, scars caused by hair removal, scars caused by glandular scleroderma, scars caused by trauma, scars caused by diabetic foot ulcers, atrophic scars, and stretch marks.

24. The pharmaceutical composition according to claim 1, wherein, The wound is a wound of the skin's epidermis; dermis; subcutaneous tissue; epidermis and dermis; dermis and subcutaneous tissue; or an injury to the epidermis, dermis, and subcutaneous tissue.

25. The pharmaceutical composition according to claim 1, wherein, The wound is selected from any one or more of the following: cut, incision, trauma, abrasion, contusion, puncture, fracture, laceration, tear, fracture, burn, and slit.

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