Composition comprising mesenchymal stem cell spheroid for treating skin wound or scar

Abstract

The present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of mesenchymal stem cell spheroids which is topically applied to a target site or administered by subcutaneous or intradermal injection for treating skin wounds or scars. Administering mesenchymal stem cell spheroids to wound or scar areas promotes dermal and subcutaneous recovery within the skin, thereby enhancing skin regeneration, scar healing, recovery, and maintenance of skin volume.

Description

Composition for treating skin wounds or scars comprising mesenchymal stem cell spheroids

[0001] The present invention relates to a composition for treating skin wounds or scars, and more particularly, to a pharmaceutical composition or filler composition for treating skin wounds or scars, comprising mesenchymal stem cell spheroids as an active ingredient, which are applied topically or administered subcutaneously or intradermally.

[0002] The skin serves as the body's first line of defense against physical and environmental aggressors, and when damaged, the wound healing process becomes activated, potentially leading to scarring. Ideally, wound healing should result in virtually scarless skin, but some individuals may experience excessive fibrosis or atrophy, resulting in keloids, hypertrophic scars, or atrophic scars. Unlike keloid and hypertrophic scars, which contain tissue outgrowth, atrophic scars produce depressed scars. The topographic depression of atrophic scars is thought to result from inadequate compensation by dermal collagen and connective tissue after injury. Atrophic scars can result from various causes, including scleroderma, stretch marks during pregnancy, acne, surgery, and accidents. Pathological scars, especially when located on visible areas of the body, can significantly reduce quality of life and cause psychological distress. The present invention proposes a novel treatment method using self-assembling adipose-derived mesenchymal stem cells (MSCs), which may contribute to improving quality of life in scar treatment.

[0003] Various treatments have been used to improve the appearance of scars. These include excision, lasers, 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 short-term fillers, lasting only a few months. Permanent fillers, such as polymethylmethacrylate (PMMA) and silicone, have long-lasting effects but carry a higher risk of complications, including granulomas, silicone embolism syndrome, and nodule formation.

[0004] To overcome the limitations of these artificial fillers, new approaches are needed, and stem cells are emerging as a new alternative. Mesenchymal stem cells (MSCs), adult stem cells, have been shown to have outstanding potential in promoting wound healing due to their self-renewal capacity, secretion of regenerative factors, and immunomodulatory properties. They can be obtained from various tissues, including bone marrow and adipose tissue. Currently, most cell therapy treatments are administered as single cells, either injected or transplanted. Single-cell MSCs have low survival rates and very low engraftment rates after transplantation. Therefore, when using MSCs as skin regeneration treatments, there is a need to improve survival and engraftment rates after transplantation. In particular, the development of cell therapy products enriched with connective tissue components is crucial for treating atrophic scars caused by the loss of connective tissue.

[0005] To address the above-mentioned issues, the inventors of the present invention established physical culture conditions optimized for skin regeneration and developed a composition containing stem cells rich in extracellular matrix and capable of engraftment and long-term volume maintenance. Furthermore, the inventors developed mesenchymal stem cell spheroids and confirmed that MSCs transplanted into the skin maintained skin volume over the long term. Through these findings, the inventors confirmed that mesenchymal stem cell spheroids overcome the limitations of existing cell-based therapies and have a more sustained effect as a dermal filler.

[0006] An object of the present invention is to provide a pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheroids that are applied topically to a target area or administered subcutaneously or intradermally.

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

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

[0009] The present invention also provides the use of mesenchymal stem cell spheroids according to the present invention for the manufacture of a medicament for treating skin wounds or scars, which is applied topically to a target site or administered subcutaneously or intradermally.

[0010] In addition, the present invention provides mesenchymal stem cell spheroids for use in treating skin wounds or scars by topically applying them to a target area or by subcutaneously or intradermally injecting them.

[0011] In addition, the present invention provides a method for treating a skin wound or scar by locally applying a therapeutically effective amount of a mesenchymal stem cell spheroid to a target area of ​​a subject in need of treatment of a skin wound or scar, or by subcutaneous or intradermal injection.

[0012] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Terms defined in commonly used dictionaries, for example, should be interpreted to have a meaning consistent with their meaning in the relevant technical context, and it is also understood that, unless explicitly defined as such herein, they will not be interpreted in an idealized or overly formal sense.

[0013] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," "the" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "including," "having," "having," "with," or variations thereof are used in either the description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0014] Unless otherwise indicated herein, references to ranges of values ​​are intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any examples or representative language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

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

[0016] The present invention relates to a pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheroids that are applied topically to a target area or administered subcutaneously or intradermally.

[0017]

[0018] Mesenchymal stem cell spheroids

[0019] The mesenchymal stem cell spheroid of the present invention can be manufactured by culturing mesenchymal stem cells (hMSCs) in two dimensions and using three-dimensional culture technology.

[0020] In one embodiment, the mesenchymal stem cell spheroids of the present invention (also referred to herein as MiB) can be prepared by isolating mesenchymal stem cells (hMSCs) from human adipose, culturing them in two dimensions, and using three-dimensional culture techniques.

[0021] The above “two-dimensional culture” means culturing cells in a monolayer in any type of culture vessel, such as a cell culture flask or flat petri dish.

[0022] The above "3D culture" is a culture in which cells interact with all three dimensions surrounding them, and has the characteristic of allowing cells to grow in all directions.

[0023] The above "mesenchymal stem cell spheroids (MiB)" refers to unit spheroids of various sizes and are a self-assembled form of cells.

[0024] In the present invention, the mesenchymal stem cell spheroid refers to a three-dimensional cell aggregate of mesenchymal stem cells produced by culturing mesenchymal stem cells, in which the cells are not dissociated but rather aggregated to form a three-dimensional cell tissue. More preferably, it is a three-dimensional cell aggregate of adipose-derived mesenchymal stem cells.

[0025] The above mesenchymal stem cell spheroid can be obtained by culturing adult stem cells, and the adult stem cells can be commercially available stem cells or those isolated from living tissue.

[0026] The above mesenchymal stem cell spheroids can effectively induce skin regeneration and healing of skin wounds and scars, and the expression of growth factors or collagen production related to skin regeneration and healing of skin wounds and scars by treatment with such mesenchymal stem cell spheroids is significantly higher than that by treatment with mesenchymal stem cell single cells (MSCs).

[0027] In the present invention, the diameter of the mesenchymal stem cell spheroid may be 10 to 800 μm, preferably 20 to 600 μm, more preferably 30 to 400 μm, still more preferably 60 to 300 μm, and still more preferably 80 to 200 μm.

[0028] In the present invention, the number of mesenchymal stem cells included in one mesenchymal stem cell spheroid 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 of mesenchymal stem cells.

[0029] In the present invention, the wet weight of the mesenchymal stem cell spheroid may 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 wet weight of the mesenchymal stem cell spheroid refers to the wet weight of one mesenchymal stem cell spheroid.

[0030] In the present invention, the dry weight of the mesenchymal stem cell spheroid may 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 mesenchymal stem cell spheroid refers to the dry weight of one mesenchymal stem cell spheroid.

[0031] In the present invention, the mesenchymal stem cell may be at least one selected from the group consisting 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. Preferably, the mesenchymal stem cell may be adipose-derived mesenchymal stem cells, and more preferably, it may be human adipose-derived mesenchymal stem cells.

[0032] In the present invention, the mesenchymal stem cell spheroid may include mesenchymal stem cells (MSCs) and an extracellular matrix (ECM). Specifically, the extracellular matrix may include collagen, fibronectin, hyaluronic acid (HA), elastin (EL), or glycosaminoglycan (GAG).

[0033] Additionally, the collagen may preferably be type 1 collagen.

[0034] More specifically, the mesenchymal stem cell spheroid according to the present invention may express collagen, fibronectin, hyaluronic acid (HA), elastin (EL), or glycosaminoglycan (GAG) at a higher level than a single mesenchymal stem cell or dermal fibroblast.

[0035] In the present invention, the mesenchymal stem cell spheroid may have increased expression of at least one selected from the group consisting 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).

[0036] Preferably, the mesenchymal stem cell spheroid may have increased expression of vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), or both.

[0037] In addition, preferably, the mesenchymal stem cell spheroid may maintain expression of fibroblast growth factor 2 (FGF2) or platelet-derived growth factor subunit A (PDGFA).

[0038] More specifically, the mesenchymal stem cell spheroid according to the present invention may have a higher expression of any one or more factors selected from the group consisting of vascular endothelial growth factor A (VEGFA), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1) compared to mesenchymal stem cells or dermal fibroblasts.

[0039] In addition, the mesenchymal stem cell spheroid according to the present invention may express fibroblast growth factor 2 (FGF2) or platelet-derived growth factor subunit A (PDGFA) at a level equivalent to that of mesenchymal stem cells or dermal fibroblasts.

[0040] In the present invention, the mesenchymal stem cell spheroid is positive for at least one marker selected from the group consisting of CD29, CD44, CD73, CD90, and CD105, and negative for at least one marker selected from the group consisting of CD31, CD45, CD79a, CD117, and HLA-DR.

[0041] The mesenchymal stem cell spheroid has cells that positively express at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of any one or more markers selected from the group consisting of CD29, CD44, CD73, CD90, and CD105. Furthermore, the mesenchymal stem cell spheroid has cells that negatively express at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2%, or less of any one or more markers selected from the group consisting of CD31, CD45, CD79a, CD117, and HLA-DR.

[0042] More specifically, the mesenchymal stem cell spheroids have cells that positively express CD29, CD44, CD73, CD90, and CD105 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. In addition, the mesenchymal stem cell spheroids have cells that negatively express CD31, CD45, CD79a, CD117, and HLA-DR by at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2%, or less.

[0043] Additionally, the mesenchymal stem cell spheroids have cells that positively express CD29, CD44, CD73, CD90, and CD105 at least 95%, 96%, 97%, 98%, or 99% or more. Additionally, the mesenchymal stem cell spheroids have cells that negatively express CD31, CD45, CD79a, CD117, and HLA-DR at least 2%, 1.5%, 1%, or less.

[0044] The mesenchymal stem cell spheroids of the present invention are derived from fat and therefore express positively the markers of CD29, CD44, CD73, CD90, and CD105, and negatively the markers of CD31, CD45, CD79a, CD117, and HLA-DR.

[0045] The mesenchymal stem cell spheroid of the present invention can express at least one protein selected from the group consisting of Tenascin (TNC), Fibronectin (FN1), Proteoglycan-4 (PRG4), COL5A3 (Collagen Type V Alpha 3 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).

[0046] Specifically, the mesenchymal stem cell spheroids of the present invention have significantly higher expression levels of tenascin (TNC) and fibronectin (FN1) compared to single mesenchymal stem cells.

[0047] In addition, the mesenchymal stem cell spheroid of the present invention specifically expresses proteoglycan (Proteoglycan-4, PRG4) and can newly 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 to single cells.

[0048] The increase in expression level mentioned above may mean, for example, 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.

[0049] In some embodiments, the mesenchymal stem cell spheroids used in the treatment are also named TRTP-101.

[0050] Composition for treating wounds or scars comprising mesenchymal stem cell spheroids

[0051] The present invention provides a pharmaceutical composition or cell therapy composition for treating skin wounds or scars comprising a therapeutically effective amount of mesenchymal stem cell spheroids.

[0052] The above pharmaceutical composition can be applied topically to an area requiring skin regeneration, specifically recovery of the entire skin layer up to the dermis or subcutaneous layer, or administered by subcutaneous or intradermal injection to treat skin wounds or scars.

[0053] In the present invention, the pharmaceutical composition can be formulated into various dosage forms such as an injection form, an infusion form, a spray form, a liquid form, a suspension form, an external preparation form, or a patch form, and preferably can be formulated into an injection form, an infusion form, a suspension form, or an external preparation form.

[0054] The above "treatment" refers to any action that improves or beneficially alters the symptoms of the disease by administering the mesenchymal stem cell spheroids according to the present invention. For example, it may provide skin regeneration or wound or scar healing in a shorter period of time compared to natural healing. The treatment may include improvement and / or relief of the wound or scar. Improvement of the wound or scar means reducing the severity of the wound or scar. Furthermore, the treatment may encompass the treatment of the wound and / or the disease associated with the wound. The treatment may refer to the healing and / or regeneration of damaged tissue resulting from the wound. The wound treatment may refer to skin regeneration, specifically, the restoration of the dermis within the skin. Furthermore, the treatment may maintain the original composition of the damaged tissue. Furthermore, the treatment may promote the healing and / or regeneration of the damaged tissue while minimizing complications and / or scarring of the wound-related disease.

[0055] In addition, the present invention provides a pharmaceutical composition for skin regeneration, skin wound or scar treatment, comprising mesenchymal stem cell spheroids; and a pharmaceutically acceptable carrier.

[0056] The pharmaceutical composition for skin regeneration, skin wound and scar treatment may include a pharmaceutically acceptable conventional inert carrier or diluent. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginate, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with a recipient to whom the composition is to be transplanted. Diluents include, but are not limited to, saline, aqueous buffers, solvents, suspending agents, and / or dispersion media. Additionally, for example, injectable formulations may further include preservatives, analgesics, solubilizers, suspending agents, or stabilizers, while topical formulations may further include bases, excipients, lubricants, suspending agents, or preservatives.In addition, in one embodiment, the suspending agent additionally included includes, but is not limited to, 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) medium, Chang's Medium, MesenCult-XF, DMEM / HG (Dulbecco's Modified Eagle's Medium high glucose) medium, or MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium low glucose). The compositions of the present invention may be used unfrozen or frozen for later use. If frozen, standard cryopreservatives (e.g., DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) may be added to the cell population before freezing. In addition, in one embodiment, the topical formulation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The topical formulation may appropriately incorporate ingredients commonly used in topical formulations such as pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed.The above-mentioned local administration preparation may also appropriately incorporate metal sequestrants such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Kalin, various herbal medicines, tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0057] The mixing ratio of the pharmaceutical composition of one specific example of the present invention can be appropriately selected depending on the type, amount, form, etc. of the additional components described above.

[0058] Method and dosage of administration of mesenchymal stem cell spheroids for treating skin wounds or scars

[0059] The present invention provides a method for treating a skin wound or scar by locally applying a therapeutically effective amount of a mesenchymal stem cell spheroid to a target area of ​​a subject requiring treatment of a skin wound or scar, or by subcutaneous or intradermal injection.

[0060] The mesenchymal stem cell spheroid of the present invention can be administered in a therapeutically effective amount to a subject in need of treatment of a skin wound or scar to regenerate skin and treat a skin wound or scar.

[0061] The mesenchymal stem cell spheroids of the present invention or pharmaceutical compositions containing them can be administered directly or indirectly using administration methods commonly used in the art. In an embodiment of the present invention, the mesenchymal stem cell spheroids can be administered subcutaneously, transdermally (application), or intradermally. Furthermore, the administration can be directly administered to a target area requiring skin regeneration through collagen production for the treatment of skin wounds or scars.

[0062] In the present invention, the target area may include a damaged or sunken area in a cell layer constituting the skin, such as the epidermis, dermis, or subcutaneous layer. Preferably, the target area may include a damaged or sunken area in the dermis, or the target area may include a damaged or sunken area in the subcutaneous layer. Specifically, damage or sunken area in the dermis may refer to damage or sunken area from the epidermis to the dermis, and damage or sunken area in the subcutaneous layer may refer to damage or sunken area in the entire skin, including the epidermis, dermis, and subcutaneous layers.

[0063] The above application is a type of percutaneous administration, and refers to any method of bringing the mesenchymal stem cell spheroid or a pharmaceutical composition containing the same into contact with a target area in an appropriate manner, thereby allowing the composition to be absorbed into the skin.

[0064] In the present invention, the mesenchymal stem cell spheroid can be administered once or multiple times.

[0065] In the present invention, the mesenchymal stem cell spheroids may be administered in a single, two, three, four, five, or more administrations. Preferably, they may be administered in one, two, or three administrations. More preferably, they may be administered in a single administration. The mesenchymal stem cell spheroids of the present invention are effective in skin regeneration and treating skin wounds or scars even with a single administration.

[0066] The above “therapeutically effective amount” means an amount sufficient to exhibit a therapeutic effect when administered to a subject in need of skin regeneration or treatment of a skin wound or scar.

[0067] In the dosage for administration to the subject, the mesenchymal stem cell spheroids are 1 to 2 × 10 in a unit dosage. 5It can include dogs, for example, the lower bound is 1 or more, or 2 or more, and the upper bound is 2 × 10 5 Dog or less, 1.8 × 10 5 Dog or less, 1.6 × 10 5 Dog or less, 1.4 × 10 5 Dog or less, 1.2 × 10 5 Dog or less, 1 × 10 5 Dog or less, 9.5 × 10 4 dog Below, 9 × 10 4 dog Below, 8.5 × 10 4 dog Below, 8 × 10 4 dog Below, 7.5 × 10 4 dog Below, 7 × 10 4 dog Below, 6.5 × 10 4 dog Below, 6 × 10 4 dog Below, 5.5 × 10 4 dog Below, 5 × 10 4 dog Below, 4.5 × 10 4 dog Below, 4 × 10 4 dog Below, 3.5 × 10 4 dog Below, 3 × 10 4 dog Below, 2.5 × 10 4 dog Below, 2 × 10 4 dog Below, 1.5 × 10 4 dog Below, 1 × 10 4 dog Below, 9.5 × 10 3 dog Below, 9 × 10 3 dog Below, 8.5 × 10 3 dog Below, 8 × 10 3 dog Below, 7.5 × 10 3 dog Below, 7 × 10 3 dog Below, 6.5 × 10 3 dog Below, 6 × 10 3 dog Below, 5.5 × 10 3 dog Below, 5 × 10 3 dog Below, 4.5 × 10 3 dog Below, 4 × 10 3 dog Below, 3.5 × 10 3 dog Below, 3 × 10 3 dog Below, 2.5 × 10 3 dog Below, or 2 × 10 3 dog Here, the number of mesenchymal stem cells included in one mesenchymal stem cell spheroid 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 of mesenchymal stem cells.

[0068] Specifically, the number of mesenchymal stem cells contained in a unit dosage is 1 × 10 2 cells to 8 × 10 7 cells, for example, the lower bound is 1.0 × 10 2 cells or more, 1.2 × 10 2 cells or more, 1.4 × 10 2 cells or more, 1.6 × 10 2 cells or more, 1.8 × 10 2 cells or more, 2.0 × 10 2cells or more, 2.2 × 10 2 cells or more, 2.4 × 10 2 cells or more, 2.6 × 10 2 cells or more, 2.8 × 10 2 cells or more, 3.0 × 10 2 cells or more, 3.2 × 10 2 cells or more, 3.4 × 10 2 cells or more, 3.6 × 10 2 cells or more, 3.8 × 10 2 cells or more, 4.0 × 10 2 cells or more, 4.2 × 10 2 cells or more, 4.4 × 10 2 cells or more, 4.6 × 10 2 cells or more, 4.8 × 10 2 cells or more, 5.0 × 10 2 cells or more, 6.0 × 10 2 cells or more, 8.0 × 10 2 cells or more, 1.0 × 10 3 cells or more, 1.2 × 10 3 cells or more, 1.4 × 10 3 cells or more, 1.6 × 10 3 cells or more, 1.8 × 10 3 cells or more, 2.0 × 10 3 cells or more, 2.2 × 10 3 cells or more, 2.4 × 10 3 cells or more, 2.6 × 10 3 cells or more, 2.8 × 10 3 cells or more, 3.0 × 10 3 cells or more, 3.2 × 10 3 cells or more, 3.4 × 10 3 cells or more, 3.6 × 10 3 cells or more, or 3.8 × 10 3cells or more, and the upper limit is 8 × 10 7 cells or less, 8.6 × 10 7 cells or less, 8.4 × 10 7 cells or less, 8.2 × 10 7 cells or less, 8 × 10 7 cells or less, 7.8 × 10 7 cells or less, 7.6 × 10 7 cells or less, 7.4 × 10 7 cells or less, 7.2 × 10 7 cells or less, 7 × 10 7 cells or less, 6.8 × 10 7 cells or less, 6.6 × 10 7 cells or less, 6.4 × 10 7 cells or less, 6.2 × 10 7 cells or less, 6 × 10 7 cells or less, 5.5 × 10 7 cells or less, 5 × 10 7 cells or less, 4.5 × 10 7 cells or less, 4 × 10 7 cells or less, 3.5 × 10 7 cells or less, 3 × 10 7 cells or less, 2.5 × 10 7 cells or less, 2 × 10 7 cells or less, 1.5 × 10 7 cells or less, 1 × 10 7 cells or less, 9 × 10 6 cells or less, 8 × 10 6 cells or less, 7 × 10 6 cells or less, 6 × 10 6 cells or less, 5 × 10 6 cells or less, 4 × 10 6 cells or less, 3 × 10 6 cells or less, 2.0 × 10 6cells, or 1.5 × 10 6 It may be less than cells.

[0069] In the present invention, the unit dosage may be 50 uL, 60 uL, 70 uL, 80 uL, 90 uL, 100 uL, 125 uL, 150 uL, 175 uL, 200 uL, 225 uL, 250 uL, 275 uL, 300 uL, 325 uL, 350 uL, 375 uL, 400 uL, 425 uL, 450 uL, 475 uL, or 500 uL.

[0070] In one embodiment, the mesenchymal stem cell spheroids are administered at a dose of 1 to 2 × 10 per unit volume of the target site. 5 It can include dogs, for example, the lower bound is 1 or more, or 2 or more, and the upper bound is 2 × 10 5 Dog or less, 1.8 × 10 5 Dog or less, 1.6 × 10 5 Dog or less, 1.4 × 10 5 Dog or less, 1.2 × 10 5 Dog or less, 1 × 10 5 Dog or less, 9.5 × 10 4 dog Below, 9 × 10 4 dog Below, 8.5 × 10 4 dog Below, 8 × 10 4 dog Below, 7.5 × 10 4 dog Below, 7 × 10 4 dog Below, 6.5 × 10 4 dog Below, 6 × 10 4 dog Below, 5.5 × 10 4 dog Below, 5 × 10 4 dog Below, 4.5 × 10 4 dog Below, 4 × 10 4 dog Below, 3.5 × 10 4 dog Below, 3 × 10 4 dog Below, 2.5 × 10 4 dog Below, 2 × 10 4 dog Below, 1.5 × 10 4 dog Below, 1 × 10 4 dog Below, 9.5 × 10 3 dog Below, 9 × 10 3 dog Below, 8.5 × 10 3 dog Below, 8 × 10 3 dog Below, 7.5 × 10 3 dog Below, 7 × 10 3 dog Below, 6.5 × 10 3 dog Below, 6 × 10 3 dog Below, 5.5 × 10 3 dog Below, 5 × 10 3 dog Below, 4.5 × 10 3 dog Below, 4 × 10 3 dog Below, 3.5 × 10 3 dog Below, 3 × 10 3 dog Below, 2.5 × 10 3 dog Below, or 2 × 10 3 dog It could be as follows:

[0071] The volume of the target area is 0.2 to 500 mm 3 may be, preferably 0.3 to 500 mm 3, 0.4 to 500 mm 3 It can be. The unit dose is administered at regular intervals depending on the area of ​​the target area. In addition, the number of mesenchymal stem cells contained in one mesenchymal stem cell spheroid 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 of mesenchymal stem cells.

[0072] Specifically, 1 × 10 based on unit dose per volume of target site 2 cells to 8 × 10 7 The number of cells can be administered, for example, the lower limit is 1.0 × 10 2 cells or more, 1.2 × 10 2 cells or more, 1.4 × 10 2 cells or more, 1.6 × 10 2 cells or more, 1.8 × 10 2 cells or more, 2.0 × 10 2 cells or more, 2.2 × 10 2 cells or more, 2.4 × 10 2 cells or more, 2.6 × 10 2 cells or more, 2.8 × 10 2 cells or more, 3.0 × 10 2 cells or more, 3.2 × 10 2 cells or more, 3.4 × 10 2 cells or more, 3.6 × 10 2 cells or more, 3.8 × 10 2 cells or more, 4.0 × 10 2 cells or more, 4.2 × 10 2 cells or more, 4.4 × 10 2 cells or more, 4.6 × 10 2 cells or more, 4.8 × 10 2cells or more, 5.0 × 10 2 cells or more, 6.0 × 10 2 cells or more, 8.0 × 10 2 cells or more, 1.0 × 10 3 cells or more, 1.2 × 10 3 cells or more, 1.4 × 10 3 cells or more, 1.6 × 10 3 cells or more, 1.8 × 10 3 cells or more, 2.0 × 10 3 cells or more, 2.2 × 10 3 cells or more, 2.4 × 10 3 cells or more, 2.6 × 10 3 cells or more, 2.8 × 10 3 cells or more, 3.0 × 10 3 cells or more, 3.2 × 10 3 cells or more, 3.4 × 10 3 cells or more, 3.6 × 10 3 cells or more, or 3.8 × 10 3 cells or more, and the upper limit is 8 × 10 7 cells or less, 8.6 × 10 7 cells or less, 8.4 × 10 7 cells or less, 8.2 × 10 7 cells or less, 8 × 10 7 cells or less, 7.8 × 10 7 cells or less, 7.6 × 10 7 cells or less, 7.4 × 10 7 cells or less, 7.2 × 10 7 cells or less, 7 × 10 7 cells or less, 6.8 × 10 7 cells or less, 6.6 × 10 7 cells or less, 6.4 × 10 7 cells or less, 6.2 × 10 7cells or less, 6 × 10 7 cells or less, 5.5 × 10 7 cells or less, 5 × 10 7 cells or less, 4.5 × 10 7 cells or less, 4 × 10 7 cells or less, 3.5 × 10 7 cells or less, 3 × 10 7 cells or less, 2.5 × 10 7 cells or less, 2 × 10 7 cells or less, 1.5 × 10 7 cells or less, 1 × 10 7 cells or less, 9 × 10 6 cells or less, 8 × 10 6 cells or less, 7 × 10 6 cells or less, 6 × 10 6 cells or less, 5 × 10 6 cells or less, 4 × 10 6 cells or less, 3 × 10 6 cells or less, 2.0 × 10 6 cells, or 1.5 × 10 6 It may be less than cells.

[0073] Here, the volume of the target area is 0.2 to 500 mm 3 may be, preferably 0.3 to 500 mm 3 , 0.4 to 500 mm 3 It can be done. The unit dose is administered at regular intervals depending on the area of ​​the target area.

[0074] In the present invention, after administration of the composition, the target area is treated or restored, enabling skin regeneration and the treatment of wounds and scars. The composition of the present invention can exhibit skin regeneration, wound and scar treatment effects for a long period of time after a single administration.

[0075] In the present invention, skin regeneration may be improving changes in the skin, such as deep or shallow wrinkles, caused by the passage of time, light, cosmetics, soap, pollution, dust, dryness, cigarettes, lifestyle habits, etc.

[0076] In the present invention, the scar may be at least one selected from the group consisting of a bedsore scar, a burn scar, a hair loss scar, a linear scleroderma scar, a wound scar, a diabetic foot ulcer scar, an atrophic scar, and a stretch mark. In addition, examples of the scar may be a scar in which the epidermis; the dermis; the subcutaneous layer; the epidermis and the dermis; the dermis and the subcutaneous layer; or the epidermis, the dermis, and the subcutaneous layer are damaged, and preferably, the scar may be a full-thickness skin scar in which the dermis is damaged, or a full-thickness skin scar in which the epidermis, the dermis, and the subcutaneous layer are damaged.

[0077] In the present invention, the wound refers to an injury to the human body caused by tissue being cut, torn, broken, burned, or traumatized, or a disorder or disease causing such damage. The wound may be an open wound or a closed wound whose surface is not open. An example of the wound may be an open wound on the skin. An example of the wound may be a wound in which the epidermis; the dermis; the epidermis and the dermis; or the epidermis, the dermis, and the subcutaneous layer are damaged, and preferably, it may be a full-thickness wound in which the dermis is damaged or a full-thickness wound in which the epidermis, the dermis, and the subcutaneous layer are damaged.

[0078] Additionally, examples of such wounds may include cuts, incisions (e.g., surgical incisions), wounds, abrasions, contusions, lacerations, lacerations, fractures, burns, or amputations.

[0079] More specifically, the mesenchymal stem cell spheroid according to the present invention may be administered directly to a scar area or wound area at a single dose, and the administration dose is as described above.

[0080] The present invention provides a filler composition for skin regeneration comprising mesenchymal stem cell spheroids.

[0081] The present invention provides the use of mesenchymal stem cell spheroids for the manufacture of a medicament for the treatment of skin wounds or scars, which is applied topically to a target site or administered subcutaneously or intradermally.

[0082] The present invention provides mesenchymal stem cell spheroids for use in treating skin wounds or scars by topically applying them to a target area or by subcutaneously or intradermally injecting them.

[0083] The present invention provides a method for treating a skin wound or scar by locally applying a therapeutically effective amount of a mesenchymal stem cell spheroid to a target area of ​​a subject requiring treatment of a skin wound or scar or by subcutaneous or intradermal injection.

[0084] The composition comprising the mesenchymal stem cell spheroid of the present invention can promote wound healing by directly administering a therapeutically effective amount to a wound, scar, or skin depression requiring dermal or subcutaneous recovery and skin regeneration, and can promote skin regeneration by inducing recovery of the dermis and subcutis within the skin, thereby promoting scar healing / recovery and skin volume maintenance.

[0085] Figure 1 shows the morphology of mesenchymal stem cells isolated from adipose tissue observed under a microscope.

[0086] Figure 2 shows the cell growth rate of mesenchymal stem cells isolated from adipose tissue.

[0087] Figure 3 shows the purity of mesenchymal stem cells confirmed by flow cytometry, and shows negative and positive markers.

[0088] Figure 4 shows the results of staining verification of the differentiation ability of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

[0089] Figure 5 shows the results of RT-PCR verification of the differentiation ability of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

[0090] Figure 6 shows microscopic images of mesenchymal stem cell spheroids, which are round cell structures of mesenchymal stem cells formed through self-assembly, at different culture days.

[0091] Figure 7 shows the size distribution of mesenchymal stem cell spheroids by donor and passage.

[0092] Figure 8 shows the cell survival of mesenchymal stem cell spheroids by donor and passage.

[0093] Figure 9 shows the DNA amount of mesenchymal stem cell spheroids according to the culture period.

[0094] Figure 10 shows an electron microscope image of a mesenchymal stem cell spheroid.

[0095] Figure 11 shows the expression of mesenchymal stem cell markers in cells constituting mesenchymal stem cell spheroids confirmed by flow cytometry.

[0096] Figure 12 shows a microscopic image of mesenchymal stem cell spheroids stained with immunofluorescence for donor-specific mesenchymal stem cell markers.

[0097] Figure 13 shows the results of quantitative RT-PCR analysis of the extracellular matrix expression level of mesenchymal stem cell spheroids.

[0098] Figure 14 shows a graph measuring extracellular matrix expression in mesenchymal stem cell spheroids using enzyme-linked immunosorbent assay.

[0099] Figure 15 shows an image confirming collagen expression in mesenchymal stem cell spheroids using fluorescent immunofluorescence staining.

[0100] Figure 16 shows an image confirming collagen expression in mesenchymal stem cell spheroids through histological analysis.

[0101] Figure 17 shows the results of analyzing the growth factor expression of mesenchymal stem cell spheroids using real-time RT-PCR.

[0102] Figure 18 shows the results of analyzing the collagen area of ​​the test group and control group that were administered mesenchymal stem cell spheroids to a wound-induced animal model. Figure 18(A) shows the results of intradermal injection of mesenchymal stem cell spheroids, and Figure 18(B) shows the results of topical administration of mesenchymal stem cell spheroids.

[0103] Figure 19 shows the results of scoring the safety and efficacy indices of the test group and control group that received intradermal administration of mesenchymal stem cell spheroids to a wound-induced animal model.

[0104] Figure 20 shows the results of analyzing the wound area ratio in the test group and control group in which mesenchymal stem cell spheroids were subcutaneously administered to a wound-induced animal model.

[0105] Figure 21 shows the results of histopathological analysis in the test group and control group in which mesenchymal stem cell spheroids were subcutaneously administered to a wound-induced animal model.

[0106] Figure 22 shows the results of analyzing the area of ​​collagen and type 1 collagen in the test group and control group administered mesenchymal stem cell spheroids to normal nude mice.

[0107] Figure 23 shows the results of dermal thickness analysis of the test group and control group administered mesenchymal stem cell spheroids to normal nude mice.

[0108] Figure 24 shows the results of an in vitro comparative analysis of the production capacity of type 1 collagen and FN in mesenchymal stem cell spheroids (TRTP-101) and HDF and MSC single cells (SC).

[0109] Figure 25 shows the results of an in vitro comparative analysis of the protein expression levels of growth factors in mesenchymal stem cell spheroids (TRTP-101) and HDF and MSC single cells (SC).

[0110] Figure 26 is a schematic diagram showing the application and recovery form of the mesenchymal stem cell spheroid of the present invention to a skin wound or scar.

[0111] Figure 27 shows Antera 3D images of atrophic scar areas before and after administration of the mesenchymal stem cell spheroid of the present invention (1 week, 4 weeks, 8 weeks). ® This shows the captured image.

[0112] Figure 28 shows the results of measuring the volume of atrophic scars before and after administration (1 week, 4 weeks, 8 weeks) of the mesenchymal stem cell spheroid of the present invention.

[0113] Figure 29 shows the deviation between experimenters in the dry weight measurement results of the mesenchymal stem cell spheroids (MiB) of the present invention.

[0114] Figure 30 shows a linear trend line for the dry weight of mesenchymal stem cell spheroids (MiB) of the present invention.

[0115] Figure 31 shows the results of the total protein count analysis of the mesenchymal stem cell spheroids and single cells of the present invention.

[0116] Figure 32 shows the results of principal component analysis of mesenchymal stem cell spheroids and single cells of the present invention.

[0117] Figure 33 shows the results of heatmap analysis of mesenchymal stem cell spheroids and single cells of the present invention.

[0118] Figure 34 shows the protein increase / decrease distribution of the mesenchymal stem cell spheroids and single cells of the present invention.

[0119] To achieve the above purpose, the present invention provides a method for producing mesenchymal stem cells separated from adipose cells into mesenchymal stem cell spheroids.

[0120] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various sizes and is not limited to the embodiments described herein.

[0121] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0122]

[0123] Example 1. Preparation and characterization of adipose-derived mesenchymal stem cell spheroids (MiB).

[0124] Example 1-1. Isolation of mesenchymal stem cells (hMSCs) from adipose tissue

[0125] Human adipose-derived mesenchymal stem cells (hMSCs), which are relatively easy to isolate and have a high growth rate, were used as the cell source for producing the mesenchymal stem cell spheroids (Microblocks, MiB) of the present invention. The isolation of hMSCs was performed with approval from the Institutional Review Board of Seoul National University Dental Hospital.

[0126] The prepared human adipose tissue was placed in Hanks® Balanced Salt solution (HBSS) containing 0.1% collagenase I and shaken at 37°C for 30 minutes to separate the cells, which were then washed and filtered through a 100 μm nylon mesh. Afterwards, the cells were supplied with nutrients from Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1X antibiotic-antimycotic solution (AA) and cultured in a 5% CO2 incubator at 37°C.

[0127] Example 1-2. Validation of Isolated Mesenchymal Stem Cells (hMSCs)

[0128] The hMSCs isolated in Example 1-1 have characteristics such as adherent morphology, multipotency, and expression of surface-specific antigens. By confirming these characteristics, it was verified that the cells isolated in Example 1-1 are hMSCs.

[0129] As a result of confirming the morphology and distribution characteristics of the above cells, the cells were found to grow by attaching to a plastic culture dish in a uniform spindle-like shape (Fig. 1), and it was confirmed that they proliferated exponentially until the 7th passage, and that it took about 2 to 3 days for the cells to divide once (Fig. 2).

[0130] In addition, to confirm whether the characteristics of hMSCs are well maintained through repeated passages, the expression of stem cell surface markers was confirmed through flow cytometry. Cultured MSCs were washed with PBS and treated with proteolytic enzymes such as trypsin and collagenase for 5 minutes at 37°C to recover single cells. The cells were reacted with fluorescently labeled antibodies against CD14, CD29, CD31, CD34, CD44, CD73, CD90, HLA-DR, CD45, CD79α, CD105, and CD117 for 1 hour at 4°C. Afterwards, the cells were washed using Dulbecco's phosphate-buffered saline (DPBS) containing 2% FBS, and the expression level compared to the isotype mouse IgG control was analyzed by FACS.

[0131] The analysis results showed that more than 95% of the cells were positive for the expression of positive markers CD29, CD44, CD73, CD90, and CD105, and less than 2% of the cells were positive for the expression of negative markers CD31, CD45, CD79a, CD117, and HLA-DR, confirming that the cells were MSCs separated with high purity (Fig. 3).

[0132] Example 1-3. Confirmation of the adipogenic differentiation potential of isolated mesenchymal stem cells (hMSCs) by staining.

[0133] To check adipocyte differentiation potential, 9.5x10 4The cells were placed in 6-well plates and cultured in a culture medium containing 0.5 mM isobutylmethylxanthine (3-isobutyl-1-methylxanthine, IBMX), 100 nM dexamethasone, 100 μM indomethacin (INDO), 10 μg / mL insulin, 10% FBS, and 1X AA in high glucose DMEM for 14 days in a 5% CO2 incubator at 37°C with the medium replaced twice a week. To confirm differentiation, the culture medium from the plates where differentiation was complete was removed, washed once with DPBS, and then fixed with 4% PFA at room temperature.

[0134] Afterwards, to visually confirm differentiation, 1 mL of a solution prepared by dissolving 0.2 g oil red O powder in 40 mL isopropanol and filtering through a 0.22 μm filter was added, reacted at room temperature for 15 minutes, and washed 5 times with DW to confirm adipogenic differentiation (Fig. 4).

[0135] Example 1-4. Confirmation of osteogenic differentiation potential of isolated mesenchymal stem cells (hMSCs) by staining.

[0136] In order to confirm the osteocyte differentiation ability of hMSCs, 9.5x10 4The cells were placed in 6-well plates and cultured in culture medium prepared by adding 10 nM dexamethasone, 50 μg / mL ascorbic acid, 10 mM β-glycerolphosphate, 1 mM dibutyryl-cAMP, 10% FBS, and 1X AA to alpha minimum essential medium (α MEM). After 4 days, the culture medium was completely removed and replaced with medium without dibutyryl-cAMP twice a week, and cultured in a 5% CO2 incubator at 37°C for 14 days to induce differentiation of hMSCs into osteocytes.

[0137] Afterwards, alizarin red S staining was performed to visually confirm osteocyte differentiation. The culture medium of the plate where differentiation was completed was removed and washed once with DPBS, and 1 mL of 4% paraformaldehyde (PFA) was added and the cells were fixed at room temperature. The fixative was removed and washed with DPBS, and 2 mL of alizarin red S solution diluted to 1% using distilled water (DW) was dispensed and reacted at room temperature for 15 minutes. After washing 5 times with DW, osteocyte differentiation (Osteogenic differentiation) was confirmed under a microscope (Olympus) while dispensed with 1 mL of DW (Fig. 4).

[0138] Example 1-5. Confirmation of the chondrocyte differentiation potential of isolated mesenchymal stem cells (hMSCs) by staining.

[0139] In order to confirm the chondrocyte differentiation ability, 9.5x10 4The cells were placed in a 6-well plate and cultured in a culture medium containing 1X insulin transferrin selenium 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). The culture medium was then added and cultured in a 5% CO2 incubator at 37°C for 21 days. Afterwards, the culture medium from the plate where differentiation was complete was removed, and the cells were fixed at room temperature using 4% PFA.

[0140] Once fixed, the cells were washed with DPBS and placed in OCT compound, frozen in an ultra-low temperature freezer (-80°C) for one day. The frozen cells were sectioned into 8 μm-thick sections using a cryocut microtome and washed with deionized water. A 1% alcian blue solution prepared using alcian blue reagent was added to the sections, stained at room temperature for 30 minutes, washed with a 0.1N HCl solution, and washed with deionized water. Chondrogenic differentiation was confirmed (Fig. 4).

[0141] Example 1-6. Verification of the differentiation potential of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes using RT-PCR.

[0142] Total RNA from cells was isolated using TRI reagent with chloroform. cDNA was synthesized from 1 μg of total RNA using Prime Script RT Master Mix. The synthesized cDNA was subjected to PCR using AccuPower PCR PreMix (Bioneer) in a PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). PCR products were analyzed by agarose gel electrophoresis (Fig. 5). The primer sets used are shown in Table 1.

[0143] [Table 1]

[0144]

[0145] As a result of confirming differentiation ability through the above Examples 1-3 to 1-6, it was confirmed that the purity of cells isolated from human fat by the method of the present invention was very high, and it was shown that they had characteristics as stem cells even during long-term culture.

[0146] Example 1-7. Production of mesenchymal stem cell spheroids (MiBs) through self-assembly of mesenchymal stem cells (MSCs).

[0147] To induce self-assembly of MSCs, MSCs cultured in an incubator were washed with PBS and treated with proteolytic enzymes such as trypsin and collagenase to recover single cells. The cells were seeded in wells, centrifuged to apply physical pressure, and then cultured three-dimensionally in an incubator at 37°C for 1, 2, 3, and 7 days. Over time, mesenchymal stem cell spheroids (also referred to herein as MiBs), which are round cell structures of a certain size, were formed (Fig. 6). Mesenchymal stem cell spheroids of a uniform size were collected by passing them through a double-stacked cell strainer with pore sizes of 70 μm and 300 μm. Mesenchymal stem cell spheroids were imaged using a JuLi stage system, and the MiB diameter was measured using ImageJ software. There was no significant difference in the diameter of mesenchymal stem cell spheroids depending on the donor and passage number, and mesenchymal stem cell spheroids with a diameter ranging from a minimum of 40 μm to a maximum of 260 μm were generated in each well, with an average diameter of 98.74 μm to 116.81 μm (Fig. 7).

[0148] Example 1-8. Cell viability of mesenchymal stem cell spheroids (MiB) by donor and passage

[0149] To investigate the live cells within the mesenchymal stem cell spheroids, cell viability was measured using the LIVE / DEAD™ Viability / Cytotoxicity Kit and the MTT Cell Proliferation Assay kit. Using the LIVE / DEAD viability / cytotoxicity kit, live cells were fluorescently stained green and dead cells red. As a result, most of the cells constituting the mesenchymal stem cell spheroids were live, while cells that were not well attached and weakly bound to the outside of the mesenchymal stem cell spheroids were stained red (Fig. 8). Compared to 2D culture, the live cell viability was 84% ​​when passage 3 MSCs were used, and there was no significant change in cell viability between passages (Fig. 8). This indicates that cells existing in a 3D environment survive more stably.

[0150] Example 1-9. Confirmation of cell growth by analyzing the DNA content of mesenchymal stem cell spheroids (MiB).

[0151] The DNA content of mesenchymal stem cell spheroids was measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit. The DNA content of mesenchymal stem cell spheroids did not change significantly during the culture period from day 1 to day 7 (Fig. 9).

[0152] These results indicate that no significant cell proliferation was detected during mesenchymal stem cell spheroid culture.

[0153] Example 1-10. Mesenchymal stem cell spheroids observed using scanning electron microscopy (SEM).

[0154] After fixation, mesenchymal stem cell spheroids were treated with HCl-EtOH solution. The treated mesenchymal stem cell spheroid samples were post-fixed with 1% osmium tetroxide, dehydrated, and chemically dried using hexamethyldisilazane (HMDS). The samples were coated with a platinum layer using Q150TS. SEM images were acquired using Apreo S at a voltage of 10 kV. As a result of observing the mesenchymal stem cell spheroids using SEM, the mesenchymal stem cell spheroids exhibited a very dense spherical structure, a very rough surface, and the formation of a large number of vesicles on the cell surface (Fig. 10).

[0155] Example 1-11. Confirmation of mesenchymal stem cell characteristics of cells constituting the manufactured mesenchymal stem cell spheroids.

[0156] To determine whether the cells constituting the mesenchymal stem cell spheroids (MiBs) still retained MSC characteristics, the MiBs were washed with PBS and dissociated into single cells by treatment with proteolytic enzymes such as trypsin and collagenase. The isolated cells were then examined for the expression of MSC surface proteins using the flow cytometry method described above.

[0157] As a result of the investigation, the cells constituting the mesenchymal stem cell spheroids expressed positive surface markers of MSCs such as CD29, CD44, CD73, CD90, and CD105 at more than 95%, and negative surface markers such as CD31, CD45, CD79a, CD117, and HLA-DR at less than 2% (Fig. 11).

[0158] Additionally, mesenchymal stem cell spheroids (MiB) were fixed and permeabilized using the Image-iT Fixation / Permeabilization Kit without disaggregation, and then incubated with antibodies against MSC markers (CD73, CD90, and CD105) in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 18 h at room temperature (RT). After washing with PBS, MiB were incubated with fluorescently labeled secondary antibodies for 6 h at RT. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at RT. It was confirmed that most cells in the fluorescently stained mesenchymal stem cell spheroids expressed the MSC markers CD73 (red), CD90 (green), and CD105 (green) (Fig. 12).

[0159] Example 1-12. Analysis of extracellular matrix expression in mesenchymal stem cell spheroids using quantitative RT-PCR.

[0160] We investigated how the expression of extracellular matrix genes changes when MSCs undergo morphological changes into mesenchymal stem cell spheroids through 3D culture using quantitative real-time RT-PCR. RNA was isolated from mesenchymal stem cells (SC) or mesenchymal stem cell spheroids (MiB) cultured for 1 and 7 days using TRI reagent as in Example 1-6, and cDNA was synthesized using Prime Script RT Master Mix. PCR reactions were performed using 2X qPCRBIO SyGreen MiX Hi-ROX. QuantiTect Primer Assays (Qiagen) were used as primers for COL1A1 (collagen type I), COL3A1 (collagen type 3), ELN (Elastin), and FN1 (fibronectin), and GAPDH was used for expression level correction. The analysis results are shown in Figure 13.

[0161] As can be seen in Figure 13, Fibronectin expression rapidly increased with the culture time of mesenchymal stem cell spheroids, and type 1 collagen also increased on the first day of culture compared to 2D cultured mesenchymal stem cells. In addition, it is expected that ECM-related gene expression increases in the mesenchymal stem cell spheroids of the present invention, and that ECM production by MSCs within the mesenchymal stem cell spheroids is possible even after administration for at least 7 days.

[0162] Example 1-13. Analysis of extracellular matrix expression in mesenchymal stem cell spheroids using enzyme-linked immunosorbent assay.

[0163] The mesenchymal stem cells or mesenchymal stem cell spheroids of the present invention were lysed with a mammalian protein extraction reagent containing 1 mM phenylmethanesulfonyl fluoride and a protease inhibitor cocktail. After centrifugation at 16,000 xg at 4°C for 20 minutes, type I collagen, fibronectin, and hyaluronic acid were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. Type I collagen, fibronectin, and hyaluronic acid were accumulated in mesenchymal stem cell spheroids (MiB) (Fig. 14).

[0164] Example 1-14. Confirmation of collagen expression in mesenchymal stem cell spheroids using fluorescent immunofluorescence staining.

[0165] To examine collagen expression in the mesenchymal stem cell spheroids (MiB) of the present invention, paraffin-embedded mesenchymal stem cell spheroids were sectioned to 4 μm thickness to produce slides. After deparaffinization and hydration of the slides, the mesenchymal stem cell spheroids (MiB) were immunostained with a fluorescently labeled type 1 collagen antibody to analyze collagen expression. The results are shown in Figure 15.

[0166] As can be seen in Figure 15, the expression of type 1 collagen was found to be significantly higher in mesenchymal stem cell spheroids (MiB) compared to single cells (Figure 15).

[0167] Example 1-15. Histological analysis confirms collagen expression in mesenchymal stem cell spheroids.

[0168] To confirm collagen expression in the mesenchymal stem cell spheroids (MiB) of the present invention through histological analysis, slides were prepared by sectioning paraffin-embedded mesenchymal stem cell spheroids into 4 μm thick sections as in Examples 1-14. After deparaffinization and hydration of the slides, immunohistochemistry (IHC) staining using a primary antibody against type 1 collagen and a biotinylated secondary antibody was performed, and it was confirmed that collagen in the mesenchymal stem cell spheroids was stained brown (Fig. 16A).

[0169] In addition, when stained using a trichrome staining kit, the area stained blue was confirmed and it was confirmed that collagen was present in the mesenchymal stem cell spheroids (Fig. 16B).

[0170] Example 1-16. Analysis of growth factor expression in mesenchymal stem cell spheroids using quantitative RT-PCR.

[0171] The growth factor expression of the mesenchymal stem cell spheroids (MiB) of the present invention was analyzed. Specifically, in order 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 in Example 1-12. Primers were designed as in Table 2, and the quantitative RT-PCR described above was performed, and the results are as in Fig. 17.

[0172] [Table 2]

[0173]

[0174] Analysis results showed that the expression of several growth factors has a positive effect on skin regeneration. As shown in Figure 17, the expression levels of FGF2 and PDGFA in mesenchymal stem cell spheroids (MiB) decreased, but the expression levels of VEGFA and HGF increased, indicating that mesenchymal stem cell spheroids contribute to angiogenesis and the regeneration of damaged tissue.

[0175] Example 2. Efficacy test using an animal model

[0176] Example 2-1. Confirmation of skin regeneration, skin wound or scar treatment effects using a wound-induced animal model.

[0177] (1) Evaluation of Masson's Trichrome (MT) collagen production ability

[0178] A full-thickness wound model was prepared by bluntly dissecting the subcutaneous tissue to remove the dermis without damaging the muscle layer and symmetrically centering on the spinal line of the rat using a scalpel. As shown in Table 3, a low dose (0.5 x 10 6 cells), medium (1.0 x 10 6 cells), high (2.0 x 10 6 TRTP-101 and TRTP-101 excipients were administered intradermally, subcutaneously, and topically, respectively, at doses of 100 cells. After protecting the wound area by attaching 3M's Tegaderm, the collagen area, etc. were checked on the 21st day.

[0179] After staining a skin tissue sample with Masson's Trichrome (MT), three different images were randomly selected from the tissue, and the collagen area in the blue area was measured using the analysis software ZEN, which was expressed as a percentage of the collagen staining area to the total area of ​​the image. The results are shown in Fig. 18. Fig. 18(A) shows the results when administered intradermally, and Fig. 18(B) shows the results when administered topically.

[0180] [Table 3]

[0181]

[0182] As can be seen in Fig. 18(A), when administered intradermally, the collagen area significantly increased by 51% in the low-dose group, which is the adipose-derived mesenchymal stem cell spheroid administration group of the present invention, and by 50% in the medium-dose group, compared to 44% in the excipient administration group. Through this, it was confirmed that statistically significantly more collagen was produced in the test group administered the adipose-derived mesenchymal stem cell spheroid of the present invention at the 21st day when the remodeling phase begins.

[0183] In addition, as can be confirmed in Fig. 18(B), when administered by the application method, the collagen area significantly increased by 53% in the low-dose group, which is the adipose-derived mesenchymal stem cell spheroid administration group of the present invention, and 50% in the high-dose group, compared to 40% in the excipient administration group, and showed a tendency to increase by 47% in the medium-dose group. Therefore, it was shown that more collagen was produced in the test group administered the adipose-derived mesenchymal stem cell spheroid of the present invention compared to the control group.

[0184] 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 wound-induced rats. As can be seen in Table 4, when comparing the normal collagen levels in rats with the collagen levels after TRTP-101 administration (unit: %), it was confirmed that the adipose-derived mesenchymal stem cell spheroids of the present invention showed a very significant tissue regeneration of up to 81%, with a skin collagen content of approximately 62% in 10-week-old normal rats (Song et al.).

[0185] [Table 4]

[0186]

[0187] (2) Evaluation of skin regeneration and wound healing when administered intradermally and subcutaneously

[0188] In order to evaluate the degree of skin regeneration and wound healing in the intradermal administration test, six indices were evaluated: four indices of inflammation level, necrosis, epithelialization, and collagenization on tissue slides stained with Hematoxylin and Eosin (H&E), and two indices of collagen fiber density and collagen fiber arrangement on MT-stained slides. The significance was evaluated by calculating the total score for each rat on a 0-5 point scale where a lower score was given as the degree of skin regeneration and wound healing improved. The results of the combined scoring of skin regeneration and wound healing indices are shown in Table 5 and Figure 19 below. Referring to Table 5 and Figure 19, it was shown that skin regeneration and wound healing significantly occurred in the medium-dose group compared to the vehicle-administered group when administered intradermally.

[0189] [Table 5]

[0190]

[0191] Each time point represents mean + SE (n=10)

[0192] * p<0.05, significant difference from the normal control group (G1) by Student's t-test

[0193]

[0194] In addition, in order to evaluate the degree of skin regeneration and wound healing in the subcutaneous administration test, the results of the wound area ratio analysis showed that TRTP-101 1.0 x 10 6 From the second to fourth day after administration, the wound area ratio in the cells / head administration group (G2) tended to be lower than that in the induced control group (G1), and in particular, on the third day, the wound area was significantly reduced compared to the vehicle administration group (Fig. 20). Based on the above results, it is thought that administration of TRTP-101 to the wound site helped with wound recovery in the early stage of wound healing.

[0195] In addition, the results of histopathological examinations scored by evaluating four indicators of inflammation level, necrosis, epithelialization, and collagenization are as shown in Figure 21. As can be seen in Figure 21, the levels of inflammation and necrosis were reduced, and it was observed that the promotion of epithelialization and collagen production in the TRTP-101 administration group (G2) administered subcutaneously increased significantly compared to the excipient administration group.

[0196] Example 2-2. Efficacy test using normal nude mice

[0197] (1) Analysis of collagen positive area and type 1 collagen positive area according to dosage

[0198] Low (0.25 x 10) levels were observed on the left and right backs of BALB / c nude mice, which lack mature T cells but are not completely immune-suppressed. 6 )(G2), medium (0.5 x 10 6 )(G3), high (1.0 x 10 6) (G4) TRTP-101 and excipient (G1) were administered intradermally once at two sites, each at 0.1 mL / site, to evaluate the persistence in the body. The results are shown in Figs. 22 and 23.

[0199] As can be seen in Fig. 22, the synthesis of collagen and type 1 collagen, which play a structural support role, was found to significantly increase in the test group administered the adipose-derived mesenchymal stem cell spheroids of the present invention compared to the control group at 4 weeks after administration (each time point represents mean + SE (n=3 to 6); *p<0.05, **p<0.01, significant difference from the excipient administration group by Student's t-test).

[0200] (2) Evaluation of in vivo persistence

[0201] As shown in Figure 23, the dermal thickness of actual mice significantly increased compared to the control group at week 8. Furthermore, the dermal layer of the medium-dose group was the thickest, and the difference was significant compared to the control group.

[0202]

[0203] Example 3. Efficacy test through in vitro experiment

[0204] Example 3-1. Confirmation of ECM production ability

[0205] In order to confirm the degree of production of ECM-related substances as in vitro efficacy, the production ability of Type I collagen and FN of adipose-derived mesenchymal stem cell spheroids (TRTP-101) of the present invention was compared with the same number of HDF (Human dermal fibroblast) and MSC single cells (SC) using ELISA analysis. The quantification results are as shown in Fig. 24.

[0206] As shown in Fig. 24, 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 fibroblast) and SC. The expression levels of collagen type I and fibronectin, which combines ECM components to form a support network, were approximately 7 times and 11 times higher, respectively, than in HDF. The adipose-derived mesenchymal stem cell spheroids of the present invention were found to produce a large amount of ECM in vitro compared to HDF (Human dermal fibroblast).

[0207] Based on these results, when the adipose-derived mesenchymal stem cell spheroid of the present invention is applied in vivo and to humans, it is expected to have an effect of building up the volume of atrophic scars through treatment and regeneration through FN, which supports skin and connective tissue by producing Collagen Type I, the largest component of dermal tissue, and promotes cell attachment, growth, and wound healing.

[0208] Example 3-2. Confirmation of growth factor secretion ability

[0209] The gene expression levels of VEGFA and HGF were analyzed using qRT-PCR, and it was confirmed that the adipose-derived mesenchymal stem cell spheroids of the present invention expressed growth factors at a higher level than HDF (Human dermal fibroblast) and MSC single cells (SC) in vitro. The results are as shown in Fig. 25.

[0210] As shown in Fig. 25, the protein expression levels of VEGF and HGF in the adipose-derived mesenchymal stem cell spheroid of the present invention were approximately 9.5 times and approximately 75 times higher, respectively. Therefore, when the adipose-derived mesenchymal stem cell spheroid of the present invention was applied to an in vivo wound model, it was confirmed that the rapid recovery observed on the third day, which is the initial stage of wound healing, was due to the high expression of growth factors related to wound healing in the adipose-derived mesenchymal stem cell spheroid of the present invention.

[0211] Example 4. Tolerance, safety, and efficacy test in patients with atrophic scars

[0212] The efficacy of TRTP-101 (adipose-derived mesenchymal stem cell spheroid) was confirmed after a single administration in patients aged 19 years or older with atrophic scars who wanted to correct the scar. 0.4 mm 3 Patients with four or more atrophic scars of abnormal volume were treated. Subjects may include patients classified as KCD disease code L90.

[0213] TRTP-101 (adipose-derived mesenchymal stem cell spheroid) was produced using fat collected from the abdomen or thigh of the test subject, and after a single administration to the subject, safety, tolerance, and efficacy were confirmed for a certain period of time.

[0214] After local anesthesia with 1% lidocaine in the patient's abdomen or thigh, 60 to 100 mL of tumescent solution (0.9% normal saline, 2% lidocaine, 0.1% epinephrine, 8.4% sodium bicarbonate) was injected using a cannula, and liposuction was performed to collect more than 60 mL of fat. After selecting and mass-producing autologous adipose-derived mesenchymal stem cells from this fat, TRTP-101 (adipose-derived mesenchymal stem cell spheroids) were cultured in three dimensions at a density of 0.5 to 4.0 × 10 7 It was manufactured so that cells / mL were present.

[0215] The cells in the syringe were gently shaken to resuspend them in the medium, and the injection site was disinfected. A single intradermal injection was administered using a 29 or 30 gauge needle. The injection was administered so that the atrophic scar area became taut, but at least 0.4 Х 10 per site 3 cells, up to 2 Х 10 6 Cells were administered.

[0216] Exploratory efficacy was analyzed for subjects who could obtain data on exploratory efficacy endpoints after administration of the clinical trial drug. Antera 3D ® Using a photocopier, photographs of the scar area were taken before and after administration of TRTP-101 (adipose-derived mesenchymal stem cell spheroids) (Fig. 27), and the volume of the atrophic scar (volume of depression) was measured. As a result, it was shown that the atrophic scar area was reduced (Fig. 28). To record the site of administration of the clinical trial drug, photographs were taken indicating the site of administration and stored together with supporting documents. The change rate before and after administration of the site of administration of the clinical trial drug was measured through the volume of the atrophic scar at each measured time point.

[0217] Referring to Figure 28, the size of the scar 8 weeks after patient administration was found to be significantly reduced compared to before administration.

[0218] To confirm tolerability and safety, no dose-limiting toxicity (DLT) corresponding to adverse drug reactions (ADRs) of Grade 3 or higher according to Common Terminology Criteria for Adverse Events (CTCAE) Ver. 5.0 occurred during the 4-week period following TRTP-101 administration. All adverse reactions were classified and analyzed by System Organ Class (SOC) and Preferred Term (PT) in the latest version of MedDRA.

[0219] The clinical trial results of Example 4, as shown in the efficacy test using an animal model, showed a significant or remarkable effect in the treatment of atrophic scars. Specifically, in the group administered TRTP-101, a pharmaceutical composition comprising adipose-derived mesenchymal stem cell spheroids of the present invention, the volume of atrophic scars was significantly reduced to a degree indicating therapeutic efficacy.

[0220]

[0221] Example 5. Confirmation of the physical properties of the mesenchymal stem cell spheroids (MiB) of the present invention.

[0222] (1) Wet weight analysis

[0223] To determine the characteristics of the mesenchymal stem cell spheroids (referred to as MiB or TRTP-101 in the present invention) manufactured in vitro according to Example 1, a wet weight analysis was performed. Specifically, to determine the weight of the mesenchymal stem cell spheroids according to the present invention, the total weight of 1,200 spheroids was measured to estimate the weight per unit, and the results are shown in Table 6 below.

[0224] [Table 6]

[0225]

[0226] As a result of the measurement, it was confirmed that the wet weight of the mesenchymal stem cell spheroid according to the present invention was approximately 3.19±0.46 μg per unit.

[0227]

[0228] (2) Dry weight analysis

[0229] In order to measure the exact weight of the mesenchymal stem cell spheroid according to the present invention, excluding the influence of solvents such as a medium or buffer, an experiment was performed to measure the dry weight using a freeze-drying method.

[0230] ①Dry weight measurement experiment workflow

[0231] The workflow of the experiment conducted to confirm the dry weight, which is one of the characteristics of mesenchymal stem cell spheroids (MiB) in vitro, is as follows. Two experimenters separately conducted the experiment on the mesenchymal stem cell spheroids (MiB) according to the present invention to confirm the variation between the experimenters. Cells were harvested when they reached 70% confluency, and 0.5K mesenchymal stem cell spheroids (MiB) were supplied with nutrients using DMEM containing 10% fetal bovine serum and 1X antibiotic and antimycotic and cultured in a 5% CO2 incubator at 37℃ for 2 days. The grouping was organized as shown in Table 7 below.

[0232] [Table 7]

[0233]

[0234] During the mesenchymal stem cell spheroid (MiB) culture period, the 5 mL tubes and parafilm used for recovery were weighed using an electronic balance before recovery. Each group was weighed three times using an electronic balance, and the average of the three measurements was calculated to calculate the weight. Before each measurement, the electronic balance was reset to zero and then reweighed. If a difference of 0.2 μg or more from the previous weight was confirmed, the zero was reset and the weight was measured again.

[0235] After 48 hours of culture, mesenchymal stem cell spheroids (MiB) were collected in 5 mL tubes in triplicate, with numbers of 0, 4,800, and 9,600, and then suspended in 200 μL of DPBS. The inlet of the 5 mL tube was sealed with parafilm, punctured with a 30-gauge needle, and dried for 2 days using a freeze-dryer. The freeze-dried samples were weighed three times using an electronic balance, and the average of the measurements was calculated to calculate the weight. The dry weights of 4,800 and 9,600 mesenchymal stem cell spheroids (MiB) were confirmed by subtracting the weights of the 5 mL tube and parafilm measured in advance from the weight of the freeze-dried samples. The calculated dry weight was then divided by the number of each mesenchymal stem cell spheroid (MiB) to obtain the weight of one mesenchymal stem cell spheroid (MiB).

[0236] ② Dry weight measurement results of mesenchymal stem cell spheroids (MiB) and mesenchymal stem cell spheroids (MiB) singlets

[0237] The dry weight of 4800 / 9600 mesenchymal stem cell spheroids (MiB) measured according to the dry weight measurement experimental workflow, and the measurement results for the dry weight of mesenchymal stem cell spheroids (MiB) are as shown in Table 8 below. The single mesenchymal stem cell spheroid (MiB) dry weight measurement deviation between the two experimenters was shown as in Figure 29, and the linear trend result for the weight of mesenchymal stem cell spheroids (MiB) is shown in Figure 30.

[0238] [Table 8]

[0239]

[0240] Referring to Table 8 and Figure 29 above, the weights of the mesenchymal stem cell spheroids (MiB) of the present invention measured by experimenters 1 and 2 were 336.2±30.5 ng and 322.9.2±17.6 ng (Mean ±SD), respectively, and there was no statistically significant difference.

[0241] In addition, the dry weight data of 0, 4800, and 9600 mesenchymal stem cell spheroids from two experimenters were integrated and used to obtain a linear trend line, and the R 2 =0.9917, indicating that the experiment was statistically significant (Fig. 30).

[0242] Additionally, the dry weight of 317 ng of one mesenchymal stem cell spheroid (MiB) that can be calculated through the trend line was found to be within the weight range of the measured mesenchymal stem cell spheroid (MiB) in Table 8.

[0243]

[0244] Example 6. Proteomic analysis of mesenchymal stem cell spheroids (MiB) of the present invention

[0245] (1) Analysis of the number of proteins constituting single cells and mesenchymal stem cell spheroids according to the present invention

[0246] In order to confirm the characteristics of the mesenchymal stem cell spheroid according to the present invention compared to single cells, a proteomic analysis of the produced mesenchymal stem cell spheroid and single cells was performed.

[0247] For this analysis, samples were placed in lysis buffer containing 4% SDS, boiled at 100°C for 10 min, and centrifuged at 18,000 rpm for 10 min to extract proteins according to the method of Lee, Jangho, et al. The proteins were measured by bicinchoninic acid assay (BCA) and separated on an SDS-PAGE gel for in-gel digestion. The gel was fractionated by molecular weight according to the method of Lee, Sang-Yeop, et al. and washed with a solution prepared with 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 tryptic digestion was performed with 50 mM ammonium bicarbonate at 37°C for 12–16 h. Tryptic peptides were extracted with a 50 mM ammonium bicarbonate solution containing 5% trifluoroacetylacid (TFA) and 50% acetonitrile and stored at 4°C.

[0248] The solution stored according to the above method was dissolved in 0.5% TFA and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS / MS). 5 μL of the eluted sample was eluted with 5%-40% acetonitrile at a flow rate of 300 nL / min for more than 95 min using a 100 μm × 2 cm nanoviper trap column and a 75 μm × 15 cm nanoviper analysis column (Thermo Fisher Scientific). All MS and MS / MS spectral data were analyzed by a QExactive Plus mass spectrometer (Thermo Fisher Scientific).

[0249] Protein identification was based on monoisotopic mass selection, a precursor mass tolerance of ±5 Da, a fragment mass tolerance of ±0.8 Da, two false cleavages, and a modified carbamidomethylcysteine. Individual spectra were accepted based on a Mascot ion threshold score of 0.05, which was specifically calculated by each database search. Peptide validator was used to match peptides and proteins exceeding the threshold, and a false discovery rate (FDR) of 1.0% was applied.

[0250] The experiment was conducted with three samples per group for single cells and mesenchymal stem cell spheroids (MiB) of the present invention, and the results are as shown in Figure 31.

[0251] As can be confirmed in Figure 31, the number of proteins constituting the single cell and the mesenchymal stem cell spheroid (MiB) of the present invention was analyzed, and it was confirmed that the total number of proteins constituting each of the mesenchymal stem cell spheroid (MiB) and the single cell slightly increased by about 3%.

[0252] (2) Principal component analysis of single cells and mesenchymal stem cell spheroids according to the present invention

[0253] To determine the correlation between groups and differences in protein composition using the proteins that make up the samples, principal component analysis (PCA) and heatmap analysis were performed. In the PCA analysis distribution graph showing the correlation of the composed proteins, it was confirmed that the single cells and mesenchymal stem cell spheroids were located oppositely, indicating a clear difference in the types of constituent proteins, and the results are as shown in Figure 32. Similarly, in the heatmap analysis showing the correlation between groups in rows, columns, and colors, differences in protein composition and content between single cells and mesenchymal stem cell spheroids were confirmed, and the results are as shown in Figure 33.

[0254] (3) Analysis of the increase / decrease distribution of proteins in mesenchymal stem cell spheroids according to the present invention

[0255] The protein increase / decrease distribution of the mesenchymal stem cell spheroids (MiB) of the present invention was analyzed compared to single cells. The protein increase / decrease distribution is shown in Figure 34, and the results confirming the increased structural proteins in the mesenchymal stem cell spheroids (MiB) of the present invention compared to single cells are shown in Table 9.

[0256] [Table 9]

[0257]

[0258] Analysis of Volcano plots (Figure 34), which visually demonstrate the statistical significance of changes in protein composition, revealed that tenascin (TNC) showed the greatest change in the mesenchymal stem cell spheroids (MiB) of the present invention compared to single cells. Tenascin is a protein expressed when tissues are damaged or inflamed, and is known to promote healing by activating the epidermal growth factor receptor (EGFR) and inflammatory signaling pathways, thereby participating in cell proliferation.

[0259] Likewise, fibronectin (FN1) is one of the proteins whose levels have been confirmed to increase significantly, and fibronectin is known to be a protein that plays a major role in wound healing and tissue remodeling processes by being involved in cell adhesion, growth, migration, and differentiation.

[0260] In this way, the proteins increased in the mesenchymal stem cell spheroid (MiB) of the present invention compared to single cells are functionally classified into structural proteins, enzymes, ribosomal proteins, histone proteins, signaling proteins, apoptosis and differentiation-related proteins, etc. In particular, proteins related to structure increased, and it is presumed that this change was due to the induction of self-assembly, which is the production principle of the mesenchymal stem cell spheroid (MiB) of the present invention.

[0261] Additionally, proteoglycan-4 (PRG4) was found to be a protein specifically expressed only in mesenchymal stem cell spheroids (MiB). PRG4 is known to promote wound healing by modulating molecular pathways associated with wound healing, inducing adipocytes to the wound site, and thereby reducing fibrosis and enhancing angiogenesis.

[0262] In addition, we confirmed that proteins that provide structural support in tissues such as skin, 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), were newly produced in mesenchymal stem cell spheroids (MiB) compared to single cells.

[0263] Through this protein analysis method, it was confirmed that several structural proteins were specifically expressed in the mesenchymal stem cell spheroids (MiB) of the present invention, and that extracellular matrix proteins also increased.

Claims

1. A pharmaceutical composition for treating skin wounds or scars, comprising a therapeutically effective amount of mesenchymal stem cell spheroids applied locally to a target area or administered subcutaneously or intradermally.

2. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid is a three-dimensional cell aggregate of mesenchymal stem cells.

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

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

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

6. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are any one selected from the group consisting 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. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid comprises mesenchymal stem cells (MSCs) and an extracellular matrix (ECM).

8. A pharmaceutical composition according to claim 7, wherein the extracellular matrix comprises at least one selected from the group consisting of collagen, fibronectin, hyaluronic acid (HA), elastin (EL), and glycosaminoglycan (GAG).

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

10. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid has increased expression of at least one growth factor selected from the group consisting 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).

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

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

13. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid comprises cells that positively express CD29, CD44, CD73, CD90, and CD105 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, and negatively express CD31, CD45, CD79a, CD117, and HLA-DR by at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2%, or less.

14. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid expresses at least one protein selected from the group consisting of Tenascin (TNC), Fibronectin (FN1), Proteoglycan-4 (PRG4), COL5A3 (Collagen Type V Alpha 3 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).

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

16. A pharmaceutical composition according to claim 1, wherein the administration is directly administered to a target area requiring skin regeneration through collagen production.

17. A pharmaceutical composition according to claim 1, wherein the target area includes a damaged or sunken portion of the dermal layer of the skin.

18. A pharmaceutical composition according to claim 1, wherein the target area includes a damaged or sunken portion of the subcutaneous layer of the skin.

19. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid induces dermal recovery within the skin and regenerates the skin.

20. A pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell spheroid induces subcutaneous recovery within the skin and regenerates the skin.

21. In the first paragraph, the pharmaceutical composition is a pharmaceutical composition in any one of the following dosage forms: injection, infusion, spray, liquid, suspension, topical, or patch.

22. In the first paragraph, the mesenchymal stem cell spheroids are 1 to 2 × 10 in the unit dosage. 5 A composition comprising a dog.

23. A pharmaceutical composition according to claim 1, wherein the scar is at least one selected from the group consisting of a scar caused by a bedsore, a scar caused by a burn, a scar caused by alopecia, a scar caused by a scleroderma, a scar caused by a wound, a scar caused by a diabetic foot ulcer, an atrophic scar, and a stretch mark.

24. A pharmaceutical composition according to claim 1, wherein the wound is a wound in which the epidermis; dermis; subcutaneous; epidermis and dermis; dermis and subcutaneous; or epidermis, dermis, and subcutaneous layers of the skin are damaged.

25. A pharmaceutical composition according to claim 1, wherein the wound is at least one selected from the group consisting of cuts, incisions, wounds, abrasions, contusions, lacerations, fractures, burns, and amputations.

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