WO2025053666A1 - Mesenchymal stem cell spheroid, preparation method therefor and composition comprising same - Google Patents

Abstract

The present invention provides a mesenchymal stem cell spheroid preparation method comprising the steps of: (a) three-dimensionally culturing mesenchymal stem cells so as to prepare spheroid cell aggregates; and (b) separating mesenchymal stem cell spheroids with an average diameter of 10-300 μm from the spheroid cell aggregates.

Description

Mesenchymal stem cell spheroid, method for producing same, and composition comprising same

The present invention relates to mesenchymal stem cell spheroids, a method for producing the same, and a composition comprising the same. Specifically, the present invention provides mesenchymal stem cell spheroids having a uniform diameter, a method for producing the same, and a composition comprising the mesenchymal stem cell spheroids and hyaluronic acid.

The skin serves as the body’s first line of defense against physical and environmental aggression, and when the skin is damaged, the wound healing process is activated, which can lead to scarring. Ideally, the wound healing process should result in virtually scarless skin, but some individuals may experience excessive fibrosis or atrophy, resulting in keloid, hypertrophic, or atrophic scars. Atrophic scars produce depressed scars, unlike keloid and hypertrophic scars that contain tissue outgrowth. The topographic depression of atrophic scars is thought to be due to inadequate compensation of dermal collagen and connective tissue following injury. Atrophic scars can result from a variety of 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 the quality of life and cause psychological stress. The present invention proposes a novel treatment method using self-assembling adipose-derived mesenchymal stem cells (MSCs) that may contribute to improving the quality of life in scar treatment.

Various treatments have been used to improve the appearance of scars. These treatments include excision, laser, and dermal filler injections. Common dermal fillers include hyaluronic acid (HA), collagen, poly-L-lactic acid, and calcium hydroxylapatite. However, hyaluronic acid and collagen are short-term fillers that only last for a few months. On the other hand, permanent fillers such as polymethylmethacrylate and silicone have a long-lasting effect but have a higher risk of complications such as granulomas, silicone embolism syndrome, and nodule formation.

To overcome the limitations of these artificial fillers, a new approach is required, and the use of stem cells is becoming a new alternative. Mesenchymal stem cells (MSCs), which are adult stem cells, have been confirmed to have excellent potential in promoting wound healing due to their self-renewal ability, secretion of factors that promote regeneration, and immunomodulatory ability. They can be obtained from various tissues such as bone marrow and adipose tissue. Currently, most cell therapy administrations are injected or transplanted in the form of single cells. Single-cell MSCs have low survival rates and very low engraftment rates when transplanted. Therefore, when using MSCs as skin regeneration therapy, it is necessary to improve them in a form with increased survival and engraftment rates after transplantation, and in particular, the development of cell therapy rich in connective tissue components is very important as a treatment for atrophic scars caused by a decrease in connective tissue.

Through this, the inventors of the present invention manufactured mesenchymal stem cell spheroids having a uniform diameter and confirmed that MSCs transplanted into the skin maintained skin volume in the long term. Through this, it was confirmed that the mesenchymal stem cell spheroids of the present invention overcome the limitations of existing cell-based therapies and have a more sustained effect as a dermal filler.

The purpose of the present invention is to provide a method for producing mesenchymal stem cell spheroids, comprising the steps of (a) producing spheroid-shaped cell aggregates by three-dimensionally culturing mesenchymal stem cells; and (b) isolating mesenchymal stem cell spheroids having an average diameter of 10 μm to 300 μm from the spheroid-shaped cell aggregates.

In addition, a mesenchymal stem cell spheroid having a uniform diameter manufactured according to the manufacturing method of the present invention is provided, which comprises mesenchymal stem cells (MSCs) and an extracellular matrix (ECM), and has an average diameter of 10 μm to 300 μm.

In addition, a composition comprising mesenchymal stem cell spheroids and hyaluronic acid manufactured according to the manufacturing method of the present invention is provided.

In addition, the present invention provides a pharmaceutical composition for skin regeneration comprising the mesenchymal stem cell spheroid.

In addition, the present invention provides a pharmaceutical composition for treating skin wounds or scars comprising the mesenchymal stem cell spheroids.

The present invention provides a pharmaceutical composition for treating skin wounds or scars, comprising mesenchymal stem cell spheroids; and a pharmaceutically acceptable carrier.

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

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.

Additionally, the present invention provides mesenchymal stem cell spheroids for use in treating skin wounds or scars.

In addition, the present invention provides a method for treating a skin wound or scar by administering a therapeutically effective amount of mesenchymal stem cell spheroids to a subject in need of treatment of a skin wound or scar.

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 art. It will also be understood that terms that are defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the relevant art, and will not be interpreted in an idealized or overly formal sense unless explicitly so defined herein.

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," and "the" are intended to include the plural forms as well, unless the context clearly indicates 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."

Unless otherwise indicated herein, references to ranges of values are merely intended to serve 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 and all 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.

The microenvironment of cells that compose mesenchymal stem cell spheroids is affected by the size of the spheroids. As the diameter increases, the supply of nutrients and oxygen to the center of the spheroid becomes limited, and this difference in the microenvironment induces changes in gene expression of mesenchymal stem cell spheroids.

Accordingly, the inventors of the present invention prepared adipose tissue-derived mesenchymal stem cell spheroids having a uniform diameter within a certain range, and confirmed that the prepared mesenchymal stem cell spheroids and a composition containing the same have excellent expression amounts or production abilities of collagen type I (ColI), fibronectin (FN1), hyaluronic acid (HA), elastin (EL), glycosaminoglycan (GAG), tenascin (TNC), and proteoglycan-4 (PRG4), and have an excellent effect in maintaining skin volume.

The mesenchymal stem cell spheroid of the present invention can be produced by culturing mesenchymal stem cells (MSCs) in two dimensions and then using a three-dimensional culture technique. A specific method for producing mesenchymal stem cell spheroids, the produced mesenchymal stem cell spheroids, and a composition containing the same will be specifically described below.

Method for producing mesenchymal stem cell spheroids

The present invention provides a method for producing mesenchymal stem cell spheroids, comprising the steps of: (a) producing spheroid-shaped cell aggregates by three-dimensionally culturing mesenchymal stem cells; and (b) isolating mesenchymal stem cell spheroids having an average diameter of 10 μm to 300 μm from the spheroid-shaped cell aggregates, to produce mesenchymal stem cell spheroids having a uniform diameter.

In the present invention, the mesenchymal stem cell spheroids rich in extracellular matrix and growth factors, uniform in size and having a controlled diameter, manufactured according to the manufacturing method of the present invention are also named MiB (Microblock).

The method for producing a mesenchymal stem cell spheroid according to the present invention comprises the step of (a) producing a spherical spheroid-shaped cell aggregate by culturing mesenchymal stem cells in three dimensions.

In one embodiment, prior to step (a), a step of isolating mesenchymal stem cells (MSCs) and then culturing them in two dimensions may be further performed.

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.

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.

The above "mesenchymal stem cell spheroid (MiB)" refers to a unit spheroid having a uniform diameter of 10 μm to 300 μm and is a self-assembled form of cells.

In the present invention, the mesenchymal stem cell spheroid is a three-dimensional cell aggregate manufactured by culturing mesenchymal stem cells, in which the cells are not dissociated but rather clumped together to exist in the form of a three-dimensional cell tissue.

The three-dimensional cell aggregate of mesenchymal stem cells may be a three-dimensional cell aggregate of any one mesenchymal stem cell selected from the group consisting of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and peripheral blood-derived mesenchymal stem cells. More preferably, it is a three-dimensional cell aggregate of adipose-derived mesenchymal stem cells.

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

In the present invention, in the step (a), 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, cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and peripheral blood-derived mesenchymal stem cells. Preferably, it may be an adipose-derived mesenchymal stem cell, and more preferably, it may be a human adipose-derived mesenchymal stem cell.

In the above step (a), the mesenchymal stem cells can 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.

In the step (a), the mesenchymal stem cells can 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.

In the above step (a), the mesenchymal stem cells can 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.

In the above step (a), the mesenchymal stem cells can positively express CD29, CD44, CD73, CD90 and CD105 at least 95%, 96%, 97%, 98% or 99% or more, or negatively express CD31, CD45, CD79a, CD117 and HLA-DR at least 2%, 1.5%, 1% or less.

Prior to the above step (a), a step of isolating mesenchymal stem cells by treating the derived tissue containing mesenchymal stem cells with a protein-decomposing enzyme such as trypsin, collagenase, papain, or elastase may be further included.

In the above step (a), mesenchymal stem cells can self-assemble to form spheroid-shaped cell aggregates.

In the present invention, (a) the step of producing a spheroid-shaped cell aggregate by culturing the mesenchymal stem cells in three dimensions can process 50 to 5,000 mesenchymal stem cells per well, for example, the lower limit may be 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, or 40 or more, and the upper limit may be 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, 800 or less, 750 or less, or 700. Below, 650 or less, or 600 or less mesenchymal stem cells can be processed.

When less than 50 mesenchymal stem cells are processed, the desired mesenchymal stem cell spheroids are not formed well, the desired size is not sufficiently achieved, and secreted factors such as extracellular matrix, which are required for spheroid formation, are not sufficiently expressed. When more than 5,000 mesenchymal stem cells are processed, there may be a problem in that cell necrosis, etc. may occur inside the mesenchymal stem cell spheroids due to the large number of cells, or it may be difficult to form mesenchymal stem cell spheroids having the desired uniform diameter.

In three-dimensional culture, 50 to 5,000 mesenchymal stem cells per well, preferably 100 to 3,000 mesenchymal stem cells per well, more preferably 150 to 2,000 mesenchymal stem cells per well, and even more preferably 200 to 800 mesenchymal stem cells per well contain a large amount of extracellular matrix (ECM), and the function of the spheroid including the mesenchymal stem cells can be maintained for a long period of time, and mesenchymal stem cell spheroids having a uniform diameter capable of maintaining high VEGF-A and HGF contents can be formed. In particular, the mesenchymal stem cell spheroids according to the present invention manufactured from a specific range of mesenchymal stem cells per well have a uniform diameter in a desired range, and thus can be suitably utilized as a filler composition for skin regeneration, a skin wound or filler treatment, etc.

In addition, the step of (a) culturing mesenchymal stem cells in three dimensions to produce a spherical spheroid-shaped cell aggregate includes a step of forming a cell aggregate by centrifuging mesenchymal stem cells.

More specifically, centrifugation treatment during three-dimensional culture of mesenchymal stem cells enables the cells to form a dense spherical structure. In particular, since the mesenchymal stem cell spheroid according to the present invention has an important cell-to-cell aggregation form, centrifugation is used to achieve cell-to-cell aggregation and maintain a uniform spherical shape while maintaining a certain level of hardness or higher.

In addition, in one embodiment, when performing the centrifugation, it is preferable that the centrifugation be performed under conditions of an acceleration of 100 to 1,000 xg, preferably 200 to 800 xg, and more preferably 300 to 600 xg for 1 to 10 minutes, preferably 2 to 8 minutes. In addition, the centrifugation can be performed at 0 to 10°C, preferably 2 to 8°C, and more preferably around 4°C.

Accordingly, the step of producing a spherical spheroid-shaped cell aggregate by culturing mesenchymal stem cells in three dimensions according to the present invention includes the step of producing a spherical spheroid-shaped cell aggregate by centrifuging 50 to 5,000 mesenchymal stem cells at 100 to 1,000 xg and culturing the same.

More specifically, the method comprises the steps of: centrifuging 50 to 5,000 mesenchymal stem cells at 100 to 1,000 xg to prepare spherical spheroid-shaped cell aggregates; and culturing the spherical spheroid-shaped cell aggregates under a culture medium for 1 to 8 days. The spherical spheroid-shaped cell aggregates can preferably be cultured under a culture medium for 1 to 5 days.

More specifically, the method comprises the steps of: centrifuging 100 to 3,000 mesenchymal stem cells at 300 to 600 xg to prepare spherical spheroid-shaped cell aggregates; and culturing the spherical spheroid-shaped cell aggregates under a culture medium for 1 to 8 days. The spherical spheroid-shaped cell aggregates can preferably be cultured under a culture medium for 1 to 5 days.

In one embodiment, the number of mesenchymal stem cells in the step of manufacturing the spheroid-shaped cell aggregate of the above-mentioned sphere may be a lower limit of 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, or 400 or more, and an upper limit may be 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, 800 or less, 750 or less, 700 or less, 650 or less, or 600.

The number of mesenchymal stem cells contained in one mesenchymal stem cell spheroid manufactured according to the present invention 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.

The term “culture medium” as used herein refers to a substance capable of supporting the growth and survival of cells in vitro .

The above medium may include, but is not particularly 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).

Additionally, the badge may contain antibiotics, antifungals and/or substances commonly used in the industry to prevent infection by bacteria, fungi and/or to prevent the growth of mycoplasma.

Specifically, the medium may contain an antibiotic such as penicillin, streptomycin or gentamicin, and preferably, an antibiotic comprising a combination of penicillin and streptomycin may be used.

In addition, the above-mentioned badge can use commonly used substances such as alporelycin B as an antifungal agent, and gentamicin, ciprofloxacin, and azithromycin as mycoplasma inhibitors, but is not limited thereto.

In addition, the medium may contain serum isolated from animals such as fetal bovine, calf, horse, sheep, pig, dog, and goat, and preferably may contain fetal bovine serum (FBS) and/or horse serum (HS). The content of FBS and/or HS contained in the medium may be 2% to 20%. The medium according to one embodiment of the present invention may contain 5%, 10%, 15%, or 20% FBS, and preferably 10% FBS. Here, % means v/v%.

A method for producing a mesenchymal stem cell spheroid according to the present invention comprises the step of (b) isolating a mesenchymal stem cell spheroid having an average diameter of 10 μm to 300 μm from the spheroid-shaped cell aggregate.

Mesenchymal stem cell spheroids can exhibit different characteristics depending on the size of the spheroid, the number of cells constituting the spheroid, etc. Accordingly, in the present invention, spheroids that contain a large amount of extracellular matrix (ECM), can maintain the function of spheroids including mesenchymal stem cells for a long period of time, and can exhibit high content of growth factors such as VEGF-A and HGF are separated into mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm.

More specifically, the method according to the present invention can pass the produced cell aggregate through a cell strainer having a diameter of 300 μm or less to produce mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm. As a result, impurities are efficiently removed, and the desired mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm are separated.

Accordingly, the step of isolating mesenchymal stem cell spheroids having an average diameter of 10 μm to 300 μm from the spheroid-shaped cell aggregates of the present invention (b) may include a step of passing the spherical spheroid-shaped cell aggregates produced in step (a) through a cell strainer having a diameter of 300 μm or less.

Specifically, the diameter of the mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm may be preferably 20 to 280 μm, more preferably 30 to 260 μm, still more preferably 40 to 240 μm, still more preferably 50 to 200 μm, and most preferably 60 to 180 μm.

The microenvironment of cells that compose mesenchymal stem cell spheroids is affected by the size of the spheroids. Therefore, when the diameter is 300 μm or more, the center of the spheroid has limited nutrient and oxygen supply, and this difference in microenvironment induces a problem of gene expression changes in the mesenchymal stem cell spheroids.

If necessary, by additionally including a strainer having a diameter larger than a certain size, cells having a diameter of mesenchymal stem cell spheroids of 10 μm to 300 μm can be secured with higher purity.

Accordingly, (b) the step of isolating cell aggregates having a uniform diameter and producing mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm may include a step of passing the spherical spheroid-shaped cell aggregates produced in step (a) through a strainer having a diameter of 300 μm or less at the upper portion and a strainer having a diameter of 10 μm or more at the lower portion together. Through this, mesenchymal stem cell spheroids having a target diameter of 10 μm to 300 μm can be produced.

The strainer having a diameter of 300 μm or less in the upper part may be, for example, a strainer having a diameter of 290 μm or less, 280 μm or less, 270 μm or less, or 260 μm or less, and preferably may be a strainer having a diameter of less than 300 μm. In addition, the strainer having a diameter of 10 μm or more in the lower part may be, for example, a strainer having a diameter of 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, or 70 μm or more.

Accordingly, the mesenchymal stem cell spheroids having an average diameter of 10 μm to 300 μm manufactured through the above manufacturing process according to the present invention contain a large amount of extracellular matrix (ECM), exhibit high ECM gene expression, and the function of the spheroids including mesenchymal stem cells can be maintained for a long period of time. In addition, they exhibit high gene expression of growth factors, and in particular, maintain high contents of VEGF-A and HGF.

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 relates to the wet weight of one mesenchymal stem cell spheroid.

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.

The mesenchymal stem cell spheroid having a diameter of 10 μm to 300 μm manufactured according to the present invention may include mesenchymal stem cells (MSCs) and an extracellular matrix (ECM). Specifically, the extracellular matrix may contain a large amount of at least one selected from the group consisting of collagen, fibronectin, hyaluronic acid (HA), elastin (EL), and glycosaminoglycan (GAG). Preferably, the collagen may be type 1 collagen.

More specifically, the mesenchymal stem cell spheroid may highly express any one or more selected from the group consisting of collagen, fibronectin, hyaluronic acid (HA), elastin (EL), and glycosaminoglycan (GAG), compared to a single mesenchymal stem cell or dermal fibroblast.

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).

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

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

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.

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.

The mesenchymal stem cells contained in the above mesenchymal stem cell spheroid can 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.

The mesenchymal stem cells contained in the above mesenchymal stem cell spheroid can negatively express at least 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2% or less for any one or more markers selected from the group consisting of CD31, CD45, CD79a, CD117 and HLA-DR.

The mesenchymal stem cells included in the above mesenchymal stem cell spheroid can 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.

The mesenchymal stem cells included in the above mesenchymal stem cell spheroid can positively express CD29, CD44, CD73, CD90 and CD105 at least 95%, 96%, 97%, 98% or 99% or more, or negatively express CD31, CD45, CD79a, CD117 and HLA-DR at least 2%, 1.5%, 1% or less.

The mesenchymal stem cell spheroids produced by the method of the present invention can express one or more proteins 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).

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.

The mesenchymal stem cell spheroids produced by the method of the present invention specifically express 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.

The increase in expression level mentioned above can mean, for example, an increase in the level of any of the proteins themselves 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.

Mesenchymal stem cell spheroids having a uniform diameter manufactured according to the manufacturing method of the present invention

The present invention provides a mesenchymal stem cell spheroid having a uniform diameter, manufactured according to the method for manufacturing the mesenchymal stem cell spheroid, comprising mesenchymal stem cells (MSCs) and an extracellular matrix (ECM), and having an average diameter of 10 μm to 300 μm.

The average diameter of the above mesenchymal stem cell spheroids may be preferably 20 to 280 μm, more preferably 30 to 260 μm, still more preferably 40 to 240 μm, still more preferably 50 to 200 μm, and most preferably 60 to 180 μm.

Each of the above mesenchymal stem cell spheroids can contain from 50 to 5,000 mesenchymal stem cells, for example, the lower limit can be 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, or 400 or more, and the upper limit can be 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 or less, 1,500 or less, 1,000 or less, 800 or less, 750 or less, 700 or less, 650 or less, or 600 mesenchymal stem cells.

The wet weight of the above 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.

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 description of the above mesenchymal stem cell spheroids is as described above in the method for producing the above mesenchymal spheroids.

Mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm according to the present invention maintain the characteristics of mesenchymal stem cells by having gene expression changes similar to the original gene expression of MSCs, while containing a large amount of extracellular matrix (ECM) such as collagen, fibronectin, hyaluronic acid, elastin or glycosaminoglycan and exhibiting high ECM gene expression. In addition, the function of spheroids including mesenchymal stem cells can be maintained for a long period of time and maintain a high content of growth factors, particularly VEGF-A and HGF.

In addition, the mesenchymal stem cell spheroids according to the present invention have high levels of gene expression of collagen types I and III. More specifically, they exhibit high expression levels of any one or more selected from the group consisting of COL1A1, COL1A2 and COL3A1.

In addition, the mesenchymal stem cell spheroids according to the present invention have high gene expression levels of collagen types (V, VI, XIII, XVIII), fibronectin and elastin. More specifically, they exhibit high expression levels of any one or more selected from the group consisting of COL5A1, COL6A1, COL13A1, COL18A1, fibronectin and elastin.

The mesenchymal stem cell spheroid according to the present invention can express any one or more proteins 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).

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.

In addition, the mesenchymal stem cell spheroids of the present invention specifically express 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.

Composition comprising mesenchymal stem cell spheroids having a diameter of 10 μm to 300 μm and hyaluronic acid

The present invention provides a composition comprising the mesenchymal stem cell spheroids and hyaluronic acid. The present invention provides a combination comprising the mesenchymal stem cell spheroids and hyaluronic acid.

The description of the above mesenchymal stem cell spheroids is as described above in the method for producing the above mesenchymal spheroids.

The composition comprises 1 to 2 × 10 of the mesenchymal stem cell spheroids.⁵It can be included in a concentration of 10/mL, for example, the lower limit is 1 or more/mL, or 2 or more/mL, and the upper limit is 2 × 10⁵Less than or equal to 1.8 × 10⁵Less than or equal to 1.6 × 10⁵Less than or equal to 1.4 × 10⁵Less than 1.2 × 10/mL⁵Less than 1 × 10⁵Less than 9.5 × 10⁴dog/mL Below, 9 × 10⁴dog/mL Below, 8.5 × 10⁴dog/mL Below, 8 × 10⁴dog/mL Below, 7.5 × 10⁴dog/mL Below, 7 × 10⁴dog/mL Below, 6.5 × 10⁴dog/mL Below, 6 × 10⁴dog/mL Below, 5.5 × 10⁴dog/mL Below, 5 × 10⁴dog/mL Below, 4.5 × 10⁴dog/mL Below, 4 × 10⁴dog/mL Below, 3.5 × 10⁴dog/mL Below, 3 × 10⁴dog/mL Below, 2.5 × 10⁴dog/mL Below, 2 × 10⁴dog/mL Below, 1.5 × 10⁴dog/mL Below, 1 × 10⁴dog Below, 9.5 × 10³dog Below, 9 × 10³dog Below, 8.5 × 10³dog Below, 8 × 10³dog Below, 7.5 × 10³dog Below, 7 × 10³dog Below, 6.5 × 10³dog Below, 6 × 10³dog Below, 5.5 × 10³dog Below, 5 × 10³dog Below, 4.5 × 10³dog Below, 4 × 10³dog Below, 3.5 × 10³dog Below, 3 × 10³dog/mL Below, 2.5 × 10³dog/mL Below, or 2 × 10³dog/mL It may be as follows. Here, the number of 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.

The composition may contain the mesenchymal stem cells at a concentration of 1 × 10 ² cells/mL to 8 × 10 ⁷ cells/mL. For example, the lower limit is 1.2 × 10 ² cells/mL or more, 1.4 × 10 ² cells/mL or more, 1.6 × 10 ² cells/mL or more, 1.8 × 10 ² cells/mL or more, 2.0 × 10 ² cells/mL or more, 2.2 × 10 ² cells/mL or more, 2.4 × 10 ² cells/mL or more, 2.6 × 10 ² cells/mL or more, 2.8 × 10 ² cells/mL or more, 3.0 × 10 ² cells/mL or more, 3.2 × 10 ² cells/mL or more, 3.4 × 10 ² cells/mL or more, 3.6 × 10 ² cells/mL or more, 3.8 × 10 ² cells/mL or more, 4.0 × 10 ² cells/mL or more, 4.5 × 10 ² cells/mL or more, 5.0 × 10 ² cells/mL or more, 6.0 × 10 ² cells/mL or more, 7.0 × 10 ² cells/mL or more, 8.0 × 10 ² cells/mL or more, 9.0 × 10 ² cells/mL or more, or 10 × 10 ² cells/mL or more, and the upper limit is 8.0 × 10 ⁷ cells/mL or less, 7.0 × 10 ⁷ cells/mL or less, 6.0 × 10 ⁷ cells/mL or less, 5.0 × 10 ⁷ cells/mL or less, 4.0 × 10 ⁷ cells/mL or less, 3.0 × 10 ⁷ cells/mL or less, 2.0 × 10 ⁷ cells/mL or less, 1.8 × 10 ⁷ cells/mL or less, 1.6 × 10 ⁷ cells/mL or less, or 1.4 × 10 ⁷ cells/mL It may be included at a concentration of less than or equal to 1.2 × 10 ⁷ cells/mL, or less than or equal to 1.0 × 10 ⁷ cells/mL.

"Hyaluronic acid (HA)" is a type of glucosaminoglycan, composed of N-acetyl glucosamine and glucuronic acid. It exists in the extracellular matrix and is involved in maintaining moisture in tissues, storing and diffusion of cell growth factors and nutrients, and is known to be synthesized by keratinocytes and fibroblasts.

The composition can comprise hyaluronic acid in a concentration of 2 to 50 mg/mL. For example, the lower limit can be at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 6 mg/mL, at least 7 mg/mL, at least 8 mg/mL, at least 9 mg/mL, at least 10 mg/mL, at least 12 mg/mL, at least 14 mg/mL, at least 16 mg/mL, at least 18 mg/mL, or at least 20 mg/mL, and the upper limit can comprise a concentration of at most 45 mg/mL, at most 40 mg/mL, at most 35 mg/mL, at most 30 mg/mL, at most 28 mg/mL, at most 27 mg/mL, or at most 26 mg/mL.

In the composition comprising the above mesenchymal stem cell spheroid and hyaluronic acid, the hyaluronic acid content may be 0.2 to 5% (w/v), and specifically, the lower limit may be 0.4% (w/v) or more, 0.6% (w/v) or more, 0.8% (w/v) or more, 1.0% (w/v) or more, 1.2% (w/v) or more, 1.4% (w/v) or more, 1.6% (w/v) or more, 1.8% (w/v) or more, 1.9% (w/v) or more, or 2% (w/v) or more, and the upper limit may be 4.5% (w/v) or less, 4.0% (w/v) or less, 3.5% (w/v) or less, 3.0% (w/v) or less, 2.8% (w/v) or less, 2.7% (w/v) or less, or It can be included in a concentration of 2.6% (w/v) or less.

The hyaluronic acid may include hyaluronic acid, a hyaluronic acid salt, or a mixture thereof. The hyaluronic acid includes pharmaceutically acceptable salts, for example, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, potassium hyaluronate, and the like.

The weight average molecular weight of the above hyaluronic acid may be 100 Da to 10,000 kDa.

The composition containing the above mesenchymal stem cell spheroid and hyaluronic acid has excellent engraftment and survival rates and excellent volume retention ability in the dermis or subcutaneous region of the skin. Due to these characteristics, it can be utilized as a tissue repair material for correcting skin loss due to aging and for correcting fine wrinkles or skin surface.

In addition, the composition containing the mesenchymal stem cell spheroid of the present invention has the effect of significantly improving the duration of volume maintenance compared to when only hyaluronic acid is administered. In addition, regeneration of surrounding tissues can be induced by various growth factors secreted from the mesenchymal stem cell spheroid according to the present invention.

Compositions, uses and methods for skin regeneration, wound or scar treatment comprising mesenchymal stem cell spheroids

The present invention provides a pharmaceutical composition for skin regeneration comprising the mesenchymal stem cell spheroid. Preferably, the pharmaceutical composition may further comprise hyaluronic acid.

The present invention provides a pharmaceutical composition or cell therapy composition for treating skin wounds or scars comprising the mesenchymal stem cell spheroids. Preferably, the cell therapy composition may further comprise hyaluronic acid.

In addition, the present invention provides a pharmaceutical composition for skin regeneration, skin wound or scar treatment, comprising mesenchymal stem cell spheroids; and hyaluronic acid.

In addition, the present invention provides a pharmaceutical composition for skin regeneration, skin wound or scar treatment, comprising mesenchymal stem cell spheroids; and hyaluronic acid.

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

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.

The pharmaceutical composition for skin regeneration, skin wound and scar treatment may contain a pharmaceutically acceptable conventional inert carrier or diluent. Pharmaceutically acceptable carriers and diluents that may be contained 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 be transplanted therewith. Diluents include, but are not limited to, saline, aqueous buffers, solvents, suspending agents, and/or dispersion media. In addition, for example, in the case of injections, preservatives, analgesics, solubilizers, suspending agents, or stabilizers may be additionally included, and in the case of topical preparations, bases, excipients, lubricants, suspending agents, or preservatives may be additionally included.

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 prior to freezing. In addition, in one embodiment, the topical formulation may be a cream, a gel, an ointment, an emulsifier, a skin suspension, a transdermal delivery patch, a drug-containing bandage, a lotion, or a combination thereof. The topical formulation may contain 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 appropriate. The above-mentioned local administration preparation may also appropriately contain metal sequestering agents such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glablidine, hot water extract of the fruit of Kalin, various crude drugs, tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and drugs such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

In the present invention, the pharmaceutical composition may 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 may be formulated into an injection form, an infusion form, a suspension form, or an external preparation form.

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.

In addition, the present invention provides a method for treating a skin wound or scar by administering a therapeutically effective amount of a mesenchymal stem cell spheroid; and hyaluronic acid to a subject in need of skin wound or scar treatment. The term "subject" of the present invention means any animal that has developed or may develop a wound or scar in the epidermal layer, dermal layer, subcutaneous layer, etc. of the skin, and typically may be an animal that can exhibit a beneficial effect by treatment using the mesenchymal stem cell spheroid of the present invention and the composition containing the same, but includes without limitation any subject that requires skin regeneration or is likely to have a skin wound or scar. As described above, the skin wound or scar can be effectively treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent, or can be administered in combination with an existing wound or scar therapeutic agent, and can be administered sequentially or simultaneously with the existing therapeutic agent.

The above "treatment" refers to any act of improving or beneficially changing the symptoms of the disease by administering the mesenchymal stem cell spheroid according to the present invention or a composition containing the same. For example, it may provide skin regeneration in a shorter period of time compared to natural healing, or healing of a wound or scar. The treatment may include improvement and/or alleviation of the wound or scar. Improvement of the wound or scar means reducing the degree of the wound or scar. In addition, the treatment may include treatment of the wound and/or a disease related to the wound. The treatment may mean healing and/or regeneration of damaged tissue caused by the wound. The wound treatment may mean skin regeneration, specifically, restoring the dermis within the skin. In addition, the treatment may maintain the original composition of the damaged tissue. In addition, the treatment may promote healing and/or regeneration of the damaged tissue while minimizing complications and/or scars of the disease related to the wound.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, it may be administered in an amount that can obtain the maximum effect with the minimum amount without causing side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount, i.e., an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prevention or treatment. The effective dosage level varies depending on the severity of the disease, the activity of the drug, the age, weight, health, and sex of the patient, the patient's sensitivity to the drug, the time of administration of the pharmaceutical composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination with or simultaneously with the pharmaceutical composition of the present invention used, and other factors well known in the medical field, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the present invention can be administered to a patient in a pharmaceutically effective amount, i.e., an amount sufficient to regenerate skin, repair and treat wounds or scars. For example, a general daily dosage can be administered in a range of about 0.0001 to 1000 mg/kg, preferably, about 0.01 to 100 mg/kg. The pharmaceutical composition of the present invention can be administered once or in several divided doses within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention can be appropriately selected by a person skilled in the art according to the administration route, administration subject, age, sex, weight, individual differences and disease state.

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

The above filler composition may additionally comprise hyaluronic acid, wherein the hyaluronic acid is as described above.

Additionally, in the filler composition, the “mesenchymal stem cell spheroid” is as described above.

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.

In addition, in the use of the above mesenchymal stem cell spheroids, the “mesenchymal stem cell spheroids” are as described above.

In addition, in the use of the above mesenchymal stem cell spheroids, “skin wound” and “skin scar” are as described above.

Additionally, the present invention provides mesenchymal stem cell spheroids for use in treating skin wounds or scars.

According to the method of the present invention, the mesenchymal stem cell spheroids have a uniform diameter and contain a large amount of extracellular matrix (ECM) such as collagen, fibronectin, hyaluronic acid, and elastin, which induce skin regeneration and wound or scar healing, or damage recovery such as skin depression, and have an excellent expression amount of growth factors, thereby promoting wound healing, and by promoting skin regeneration through inducing dermal recovery within the skin, they can promote scar healing/recovery and skin volume maintenance.

Figure 1 shows a method for producing mesenchymal stem cell spheroids rich in extracellular matrix and growth factors.

Figure 2 shows the morphology of mesenchymal stem cells isolated from adipose tissue observed under a microscope.

Figure 3 shows the cell growth rate of mesenchymal stem cells isolated from adipose tissue.

Figure 4 shows the purity of mesenchymal stem cells confirmed by flow cytometry, and shows negative and positive markers.

Figure 5 shows the results of staining verification of the differentiation ability of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

Figure 6 shows the results of RT-PCR verification of the differentiation ability of mesenchymal stem cells into adipocytes, osteocytes, and chondrocytes.

Figure 7 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.

Figure 8 shows the size distribution of mesenchymal stem cell spheroids by donor and passage.

Figure 9 shows the cell survival of mesenchymal stem cell spheroids by donor and passage.

Figure 10 shows the DNA amount of mesenchymal stem cell spheroids according to the culture period.

Figure 11 shows an electron microscope image of a mesenchymal stem cell spheroid.

Figure 12 shows the expression of mesenchymal stem cell markers in cells constituting mesenchymal stem cell spheroids confirmed by flow cytometry.

Figure 13 shows a microscopic image of donor-specific mesenchymal stem cell markers in immunofluorescence staining of mesenchymal stem cell spheroids.

Figure 14 shows the results of quantitative RT-PCR analysis of the extracellular matrix expression level of mesenchymal stem cell spheroids.

Figure 15 shows a graph measuring extracellular matrix expression in mesenchymal stem cell spheroids using enzyme-linked immunosorbent assay.

Figure 16 shows an image confirming collagen expression in mesenchymal stem cell spheroids by fluorescent immunohistochemistry staining.

Figure 17 shows an image confirming collagen expression in mesenchymal stem cell spheroids by histological analysis.

Figure 18 shows the results of analyzing the growth factor expression of mesenchymal stem cell spheroids using real-time RT-PCR.

Figure 19 shows the results of analyzing the effect of the diameter size of mesenchymal stem cell spheroids on gene expression.

Figure 20 shows the results of comparative analysis of skin extracellular matrix gene expression levels in mesenchymal stem cell spheroids of different sizes.

Figure 21 shows the volume retention ability of a composition comprising mesenchymal stem cell spheroids and hyaluronic acid injected into mouse skin.

Figure 22 is a histological image showing the intradermal retention of a composition containing mesenchymal stem cell spheroids and hyaluronic acid injected into a mouse.

Figure 23 shows the survival and engraftment ability of a composition comprising mesenchymal stem cell spheroids and hyaluronic acid injected into mice.

Figure 24 shows an image of blood vessels surrounding a composition containing mesenchymal stem cell spheroids and hyaluronic acid injected into a mouse, analyzed by immunohistochemical staining.

Figure 25 shows the deviation between experimenters in the dry weight measurement results of the mesenchymal stem cell spheroids (MiB) of the present invention.

Figure 26 shows a linear trend line for the dry weight of mesenchymal stem cell spheroids (MiB) of the present invention.

Figure 27 shows the results of the total protein count analysis of the mesenchymal stem cell spheroids and single cells of the present invention.

Figure 28 shows the results of principal component analysis of mesenchymal stem cell spheroids and single cells of the present invention.

Figure 29 shows the results of heat map analysis of mesenchymal stem cell spheroids and single cells of the present invention.

Figure 30 shows the protein increase/decrease distribution of the mesenchymal stem cell spheroids and single cells of the present invention.

To achieve the above purpose, the present invention provides a method for producing mesenchymal stem cells into mesenchymal stem cell spheroids having a uniform size and a pharmaceutical use thereof.

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

The present invention will be described in more detail through the following examples; however, the following examples are for the purpose of explanation only and are not intended to limit the scope of the present invention.

Example 1. Production and characterization of adipose tissue-derived mesenchymal stem cells (MSCs)

Example 1-1. Isolation of mesenchymal stem cells (MSCs) from adipose tissue

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

The prepared human-derived 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 tissue was cultured in a 5% CO ₂ incubator at 37°C, supplying nutrients with Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) and 1X antibioticantimycotic solution (AA).

Example 1-2. Verification of Isolated Mesenchymal Stem Cells (MSCs)

The MSCs isolated according to Example 1-1 have characteristics such as an attachment form, multipotency, and expression of surface-specific antigens, and by confirming these characteristics, it was verified that the cells isolated in Example 1-1 were MSCs.

As a result of confirming the morphological and distribution characteristics of the above cells, the cells were found to attach and grow in a homogeneous spindle-like shape on a plastic culture dish (Fig. 2), and were found to proliferate exponentially up to the 7th passage, and it was confirmed that it takes about 2 to 3 days for the cells to divide once (Fig. 3).

In addition, to confirm whether the characteristics of MSCs 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 at 37℃ for 5 minutes to recover single cells. The cells were reacted with antibodies to fluorescently labeled CD14, CD29, CD31, CD34, CD44, CD73, CD90, HLA-DR, CD45, CD79α, CD105, and CD117 for 1 hour at 4℃. 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 group was analyzed by FACS.

The test 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% for the expression of negative markers CD31, CD45, CD79a, CD117, and HLA-DR, confirming that the cells were MSCs separated with high purity (Fig. 4).

Example 1-3. Confirmation of adipocyte differentiation potential of isolated mesenchymal stem cells (MSC) by staining

To confirm the adipogenic differentiation potential of the isolated mesenchymal stem cells, 9.5 x 10 ⁴ cells were plated in a 6-well plate and cultured in a culture medium prepared with high glucose DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 100 nM dexamethasone, 100 μM indomethacin (INDO), 10 μg/mL insulin, 10% FBS, and 1X AA, with the medium replaced twice a week in a 5% CO ₂ incubator at 37°C for 14 days. To confirm differentiation, the culture medium from the plate where differentiation was completed was removed, washed once with DPBS, and then fixed with 4% PFA at room temperature.

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

Example 1-4. Confirmation of osteocyte differentiation potential of isolated mesenchymal stem cells (MSC) by staining

To confirm the osteocyte differentiation potential of isolated mesenchymal stem cells (MSCs), 9.5 x 10 ⁴ cells were seeded in a 6-well plate using the same method as in Example 1-3 and cultured in a 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 (αMEM). After 4 days, the culture medium was completely removed and replaced with a medium without dibutyryl-cAMP twice a week, and cultured in a 5% CO ₂ incubator at 37°C for 14 days to induce differentiation of MSCs into osteocytes.

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, 1 mL of DW was dispensed and osteocyte differentiation (osteogenic differentiation) was confirmed under a microscope (Olympus) (Fig. 5).

Example 1-5. Confirmation of the chondrocyte differentiation potential of isolated mesenchymal stem cells (MSCs) by staining

To confirm the chondrocyte differentiation potential of the isolated mesenchymal stem cells (MSCs), 9.5 x 10 ⁴ cells were placed in a 6-well plate using the same method as in Example 1-3 and cultured in a culture medium prepared by adding 1X insulin transferrin selenium premix (ITS), 50 ng/mL ascorbic acid, 40 μg/mL L-proline, 100 nM dexamethasone, 10% FBS, and 1X AA to αMEM, and 10 ng/mL transforming growth factor (TGF-β3) was added. The cells were cultured in a 5% CO ₂ incubator at 37°C for 21 days. Thereafter, the culture medium of the plate where differentiation was completed was removed, and the cells were fixed at room temperature using 4% PFA.

Once the cells were fixed, they were washed with DPBS and the fixed cells were placed in OCT compound and frozen in an ultra-low temperature freezer (-80℃) for one day. The frozen cells were sectioned into 8 μm thick sections using a cryocut microtome and washed with DW. A 1% alcian blue solution prepared using an alcian blue reagent was added to the sections, stained for 30 minutes at room temperature, washed with a 0.1N HCl solution, and washed with DW to confirm differentiation into chondrocytes (Chondrogenic differentiation) (Fig. 5).

Example 1-6. Verification of differentiation potential into adipocytes, osteocytes, and chondrocytes, which are characteristics of mesenchymal stem cells (MSCs), using RT-PCR

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 reaction using AccuPower PCR PreMix (Bioneer) in a PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were analyzed by agarose gel electrophoresis (Fig. 6). The primer sets used are as shown in Table 1.

[Table 1]

As a result of confirming differentiation ability through the above Examples 1-3 to 1-6, it was confirmed that the purity of mesenchymal stem 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 (Fig. 6).

Example 2. Production and characterization of adipose-derived mesenchymal stem cell spheroids (MiB)

Example 2-1. Production of mesenchymal stem cell spheroids (MiB) through self-assembly of mesenchymal stem cells (MSCs)

For self-assembly of the isolated mesenchymal stem cells (MSCs) of Example 1-1, the MSCs cultured in the incubator were washed with PBS and treated with protein-decomposing enzymes such as trypsin and collagenase to recover single cells. 100 to 3,000 cells were seeded in a well, centrifuged to apply physical pressure, and then three-dimensionally cultured in a 37°C incubator for 1, 2, 3, and 7 days. Over time, mesenchymal stem cell spheroids (also referred to herein as MiB), which are round cell aggregates of a certain size, were generated (Fig. 7).

Thereafter, the mesenchymal stem cell spheroids were passed through a double-stacked cell strainer with pore sizes of 70 μm and 300 μm to isolate mesenchymal stem cell spheroids with a uniform diameter. That is, by passing through the above double-stacked cell strainer, spheroids with sizes of 70 μm to 300 μm were classified, and these spheroids had a uniform spherical shape.

The above mesenchymal stem cell spheroids were imaged using a JuLi stage system, and the MiB diameter was measured using ImageJ software. The diameter of the mesenchymal stem cell spheroids did not differ significantly depending on the donor and passage number, and mesenchymal stem cell spheroids with a diameter of at least 40 μm and a maximum of 260 μm were generated in each well, and the average diameter was 98.74 μm to 116.81 μm, showing a uniform diameter (Fig. 8).

Example 2-2. Production of mesenchymal stem cell spheroids (MiB) through self-assembly of mesenchymal stem cells (MSCs)

For self-assembly of the isolated mesenchymal stem cells (MSCs) of Example 1-1, the MSCs cultured in the incubator were washed with PBS and treated with protein-decomposing enzymes such as trypsin and collagenase to recover single cells. 100 to 2,500 cells were seeded in a well, centrifuged to apply physical pressure, and then three-dimensionally cultured in a 37°C incubator for 1, 2, 3, and 7 days. Over time, round mesenchymal stem cell spheroids (also referred to herein as MiB) of a constant size were generated, with an average diameter of 98.74 μm to 116.81 μm.

Example 2-3. Cell viability by donor and passage of mesenchymal stem cell spheroids (MiB)

In order to investigate living cells in the mesenchymal stem cell spheroids manufactured by the method of the present invention, cell viability was measured using the LIVE/DEAD ^TM Viability/Cytotoxicity Kit and the MTT Cell Proliferation Assay kit. As a result of fluorescently staining living cells in green and dead cells in red through LIVE/DEAD viability/cytotoxicity kit staining, most of the cells constituting the mesenchymal stem cell spheroids were alive, and cells that were not well bound and weakly bound to the outside of the mesenchymal stem cell spheroids were stained red (Fig. 9). Compared to when cultured in two dimensions, the living cells showed a viability of 84% when passage 3 MSCs were used, and there was no significant change in cell viability between passages (Fig. 9). This indicates that cells existing in a three-dimensional environment survive more stably.

Example 2-4. Confirmation of cell growth by analyzing DNA content of mesenchymal stem cell spheroids (MiB)

The DNA content of mesenchymal stem cell spheroids manufactured according to the method of the present invention was measured using Quant-iT ^TM PicoGreen ^TM 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. 10).

These results demonstrate that no significant cell proliferation is detected during the culture of mesenchymal stem cell spheroids, and that the cells remain highly viable for a certain period of time.

Example 2-5. Mesenchymal stem cell spheroids observed by scanning electron microscopy (SEM)

After fixing the mesenchymal stem cell spheroids, they 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). They 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 a large number of vesicles formed on the cell surface (Fig. 11). Through these results, it was confirmed that a large amount of extracellular vesicles rich in bioreactive materials were formed when the mesenchymal stem cell spheroids were produced according to the manufacturing method of the present invention, and that they are effective for wound healing, skin regeneration, and immune regulation.

Example 2-6. Confirmation of mesenchymal stem cell characteristics of cells forming mesenchymal stem cell spheroids

In order to confirm whether the cells constituting the mesenchymal stem cell spheroids manufactured according to an embodiment of the present invention still maintain the characteristics of MSCs, the mesenchymal stem cell spheroids were washed with PBS and separated into single cells by treating them with protein-decomposing enzymes such as trypsin and collagenase. The separated cells were examined for the expression of mesenchymal stem cell surface proteins using the flow cytometry method described above. The cells constituting the mesenchymal stem cell spheroids expressed positive surface markers of MSCs such as CD29, CD44, CD73, CD90, and CD105 by more than 95%, and negative surface markers such as CD31, CD45, CD79a, CD117, and HLA-DR by less than 2% (Fig. 12).

In addition, after fixation and membrane permeability were increased using the Image-iT Fixation/Permeabilization Kit without disaggregating MiB, the cells were incubated with antibodies against MSC markers (CD73, CD90, CD105) in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 18 h at room temperature (RT). After washing with PBS, MiB was incubated with fluorescently labeled secondary antibodies for 6 h at RT. Nuclei (blue) were stained by treating with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at RT. As a result, 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. 13).

Example 2-7. Analysis of the expression level of extracellular matrix in mesenchymal stem cell spheroids using quantitative RT-PCR

Quantitative real-time RT-PCR (qRT-PCR) was used to investigate how the expression of extracellular matrix genes changes when MSCs undergo morphological changes into mesenchymal stem cell spheroids through 3D culture. 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 Fig. 14.

As can be confirmed in Fig. 14, 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 in mesenchymal stem cell spheroids is possible even after administration for at least 7 days.

Example 2-8. Analysis of extracellular matrix expression of mesenchymal stem cell spheroids using enzyme-linked immunosorbent assay

In order to analyze the amount of extracellular matrix expression of the mesenchymal stem cells or mesenchymal stem cell spheroids according to the present invention, the mesenchymal stem cells or mesenchymal stem cell spheroids according to 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 ×g at 4°C for 20 minutes, type 1 collagen, fibronectin, and hyaluronic acid were analyzed using an ELISA (Enzyme-linked immunosorbent assay) kit. As a result, it was found that type 1 collagen, fibronectin, and hyaluronic acid were accumulated in the mesenchymal stem cell spheroids (MiB) (Fig. 15).

Example 2-9. Confirmation of collagen expression in mesenchymal stem cell spheroids by fluorescent immunohistochemistry

The expression of collagen, an extracellular matrix component included in the mesenchymal stem cell spheroid (MiB) according to the present invention, was confirmed. Specifically, the paraffin-embedded mesenchymal stem cell spheroid was cut to a thickness of 4 μm to produce a slide. After deparaffinization and hydration of the slide, the mesenchymal stem cell spheroid (MiB) was immunostained with a fluorescently labeled type 1 collagen antibody to analyze collagen expression, and the results are as shown in Fig. 16.

As can be seen in Figure 16, the expression of type 1 collagen was found to be significantly higher in mesenchymal stem cell spheroids (MiB) compared to single cells.

Example 2-10. Confirmation of collagen expression in mesenchymal stem cell spheroids by histological analysis

Collagen expression of the mesenchymal stem cell spheroids according to the present invention was confirmed by histological analysis. Specifically, the paraffin-embedded mesenchymal stem cell spheroids were sectioned to a thickness of 4 μm as in Example 2-9 to produce slides. After deparaffinization and hydration, the slides were stained with immunohistochemistry (IHC) using a primary antibody against type 1 collagen and a biotinylated secondary antibody.

As a result, it was confirmed that collagen was stained brown in the mesenchymal stem cell spheroids (Fig. 17A). 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. 17B).

Example 2-11. Analysis of growth factor expression in mesenchymal stem cell spheroids using quantitative RT-PCR

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 mesenchymal stem cell spheroids (MiB) manufactured according to the method of the present invention, quantitative RT-PCR was performed as in Example 2-7. Primers were designed as shown in Table 2, and the quantitative RT-PCR described above was performed, and the results are as shown in Fig. 18.

[Table 2]

The analysis results showed that the expression of several growth factors had a positive effect on skin regeneration. As shown in Fig. 18, 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 the formation of new blood vessels and the regeneration of damaged tissue.

Example 2-12. Analysis of gene expression differences and extracellular matrix expression levels according to the diameter size of mesenchymal stem cell spheroids

To investigate the differences in gene expression according to the diameter size of mesenchymal stem cell spheroids, 500 and 3,000 cells were seeded per well, respectively. Two types of mesenchymal stem cell spheroids with diameters of approximately 100 μm and approximately 300 μm were prepared and RNA was isolated.

Specifically, an mRNA sequencing library was synthesized using the Illumina Truseq Stranded mRNA/total Library prep kit, and clusters were constructed using the cBot cluster generation automation system, and then the sequence of each amplified cDNA was sequenced using the Novaseq 6000 sequencing system (Illumina). The gene expression of two types of mesenchymal stem cell spheroids with different diameters was mapped using Tophat (v2.0.13), and the differentially expressed gene (DEG) was analyzed using Cuffdiff (v2.2.0). Genes showing significant expression changes were selected and analyzed using DAVID for gene ontology to investigate the pathway affected by the spheroid size, and the results are shown in Figs. 19 and 20.

As shown in the heatmap of Fig. 19, compared to the two-dimensionally cultured mesenchymal stem cells (SC), the number of genes showing expression changes in the mesenchymal stem cell spheroids of the present invention increased as the diameter increased. The number of genes whose expression increased by more than 2-fold was 556 for the 100 μm diameter mesenchymal stem cell spheroids and 787 for the 300 μm diameter mesenchymal stem cell spheroids, and the number of genes whose expression decreased was 816 for the 100 μm diameter spheroids and 898 for the 300 μm diameter mesenchymal stem cell spheroids. In addition, the 100 μm diameter mesenchymal stem cell spheroids showed high ECM organization and cell adhesion in terms of Biological process, and high ECM components and ECM space in terms of Cellular Components. In addition, in terms of molecular function, the characteristics were high in the following order: ECM binding, structural constituent of cytoskeleton, collagen binding, heparin binding, growth factor activity, and ECM structural constituent.

These results suggest that the microenvironment of cells composing mesenchymal stem cell spheroids is affected by the size of the spheroids, and that as the diameter increases, differences in the microenvironment, such as limited supply of nutrients and oxygen to the center of the mesenchymal stem cell spheroid, induced changes in gene expression in 300 μm mesenchymal stem cell spheroids compared to 100 μm mesenchymal stem cell spheroids, showing differences from the unique gene expression pattern of mesenchymal stem cells.

In addition, the expression of genes constituting the extracellular matrix was much higher in 100 μm mesenchymal stem cell spheroids than in 300 μm mesenchymal stem cell spheroids. Among the extracellular matrix components, collagen is a major protein that accounts for 77% of the fat-free dry weight of skin tissue, excluding fat and water, and is very important for the strength and elasticity of the skin. There are 28 types of collagen, and collagen type I accounts for 80-90% of skin collagen, and type III accounts for 10-15%.

Accordingly, as a result of comparing the gene expression of the extracellular matrix in each mesenchymal stem cell spheroid with a diameter of 100 μm and 300 μm, it was confirmed that the gene expression of collagen types I and III was much higher in the mesenchymal stem cell spheroid with a diameter of 100 μm, as can be confirmed in Fig. 20 (Fig. 20A). In addition, the gene expression of other collagen types (V, VI, XIII, XVIII), fibronectin, and elastin was found to be higher in the mesenchymal stem cell spheroid with a diameter of 100 μm (Fig. 20B).

Therefore, mesenchymal stem cell spheroids have different gene expression levels depending on their diameter, and it was confirmed that mesenchymal stem cell spheroids having a specific range of diameters manufactured according to the present invention exhibit very high expression of ECM genes and are highly suitable for treating skin wounds or scars.

Example 3. Mesenchymal stem cell spheroid-hyaluronic acid (HA) composition (MiB-HA) and its characteristics

Example 3-1. Preparation of mesenchymal stem cell spheroid-hyaluronic acid (HA) composition (MiB-HA)

Mesenchymal stem cell spheroids (MiB) manufactured according to the present invention (2.5x10 ⁵ or ^2.5x106 cells/mL) was evenly distributed in 2 mg/mL of hyaluronic acid (HA) to prepare a mesenchymal stem cell spheroid-hyaluronic acid composition. The administered dose was 0.1 mL.

Example 3-2. Volume maintenance ability of mesenchymal stem cell spheroids injected into mouse skin

The volume was maintained by evaluating the mesenchymal stem cell spheroids manufactured according to the present invention to determine the sustainability and side effects.

Specifically, 7-week-old non-obese diabetic SCID gamma (NSG) mice [NOD, Cg-Prkdcscid Il2rg tm 1Wjl/SzJ] were randomly divided into four groups as shown in Table 3 below.

[Table 3]

SA, dHA, and mesenchymal stem cell spheroid-hyaluronic acid composition (MiB-HA) were injected subcutaneously into two left and two right areas of the dorsal skin, and the length, width, and height of the injected skin were measured weekly using a caliper for 12 weeks to measure the residual volume, and the volume was calculated. The volume was calculated using the following equation.

Volume = 4/3 x π x (1/2 x length) x (1/2 x width) x (1/2 x height)

As can be seen in Fig. 21, when a composition containing low-dose (Low) and high-dose (High) mesenchymal stem cell spheroids and hyaluronic acid was injected, the initial volume was well maintained for up to 4 weeks after injection. The volume of both doses of mesenchymal stem cell spheroids decreased slightly over time from 5 weeks after injection, but the high-dose mesenchymal stem cell spheroids (High) were found to significantly maintain the volume compared to the dHA group for up to 12 weeks.

In addition, for histological analysis, the skin was collected 8 or 12 weeks after injection of the composition containing mesenchymal stem cell spheroids and hyaluronic acid, fixed with 4% PFA, and embedded in paraffin. Tissue slides sectioned at 4 μm thickness were deparaffinized and stained using a Trichrome Staining Kit. As a result, unlike the dHA transplantation that disappeared 8 weeks after transplantation, the composition containing mesenchymal stem cell spheroids (Low, High) and hyaluronic acid was observed to remain in the subcutaneous area of the mouse and maintain the volume (Fig. 22).

Example 3-3. Engraftment and viability of mesenchymal stem cell spheroid (Microblock)-hyaluronic acid (HA) composition injected into mouse skin

To confirm the survival of the transplanted mesenchymal stem cell spheroids, PCR using human-specific Alu sequences and SISH for human-specific HER2 were performed. After transplanting the composition containing mesenchymal stem cell spheroids and hyaluronic acid, the mice were sacrificed 4 or 8 weeks later, the skin was cut, and DNA was extracted using AccuPrep Genomic DNA Extraction Kit. Using the primers in Table 4 below, PCR was performed at 94°C for 5 minutes, 94°C for 30 seconds, 58°C for 15 seconds, and 72°C for 30 seconds, 35 cycles. The PCR products were confirmed by electrophoresis through 1% agarose gel.

[Table 4]

PCR analysis results showed that human-specific Alu sequences, which are expressed only in human cells, were expressed in tissue samples from mice 4 and 8 weeks after transplantation of compositions containing mesenchymal stem cell spheroids (Figure 23A).

In addition, silver-enhanced in situ hybridization (SISH) was performed to re-verify the detection of human cells transplanted into mouse skin. Specifically, deparaffinized tissue slides were denatured by treating with citrate, and the human epidermal growth factor receptor 2 (HER2) and chromosome 17 probe were mixed and reacted in a hybridization solution. After washing, the HER2 binding signal was visualized by silver precipitation after sequential addition of silver acetate, hydroquinone, and H ₂ O _{2 ,} and the chromosome 17 signal was visualized by reaction with fast red and naphthol phosphate. Visualized by counterstaining with hematoxylin, the injected mesenchymal stem cell spheroids were viable under the skin of mice 4 weeks after transplantation (Fig. 23B). Fluorescent immunohistochemistry using fluorescently labeled STEM 101 antibody that specifically identifies the nucleus of human cells also confirmed the successful engraftment and survival of the injected mesenchymal stem cell spheroids into the skin (Fig. 23C).

Example 3-4. Analysis of blood vessels surrounding the engraftment of mesenchymal stem cell spheroids injected into mouse skin by immunostaining

Given the multilineage differentiation potential of MSCs, including the ability to differentiate into endothelial cells, it is important to discern whether the endothelial cells originate from mouse angiogenesis or human mesenchymal stem cell differentiation.

To stain blood vessels, the skin was collected 8 weeks after the injection of mesenchymal stem cell spheroids as in Example 3-2, fixed with 4% PFA, and embedded in paraffin. Tissue slides were serially sectioned at 4 μm thickness, and CD31 primary antibody or XRCC5 primary antibody was diluted and fixed, and the Discovery XT automated immunohistochemical stainer was used. Detection was performed using Ventana ChromoMap Kit, and the sections were visualized after counterstaining with hematoxylin and bluing reagent. XRCC5 enables specific detection of human nuclei. XRCC5 was not detected in CD31-positive microvessels at the site of engraftment of mesenchymal stem cell spheroids (Fig. 24). It was confirmed that the microvessels found in the periphery where the injected mesenchymal stem cell spheroids engrafted were not differentiated from human mesenchymal stem cells but derived from adjacent mouse blood vessels.

Example 4. Confirmation of physical characteristics of mesenchymal stem cell spheroids (MiB) of the present invention

(1) Wet weight analysis

In order to confirm the in vitro characteristics of the mesenchymal stem cell spheroids manufactured according to the present invention (referred to as MiB or TRTP-101 in the present invention), a wet weight analysis was performed. Specifically, in order to confirm the weight of the mesenchymal stem cell spheroids according to the present invention, the total weight of 1,200 pieces, which is the production quantity, was measured to estimate the weight per piece, and the results are as shown in Table 5 below.

[Table 5]

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

(2) Dry weight analysis

In order to measure the exact weight of the mesenchymal stem cell spheroids 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.

①Dry weight measurement experiment workflow

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. The cells were harvested when they were 70% confluent, and the 0.5K mesenchymal stem cell spheroids (MiB) were cultured in a 5% CO ₂ incubator at 37°C for 2 days while supplying nutrients with DMEM containing 10% fetal bovine serum and 1X antibiotic and antimycotic. The grouping was configured as shown in Table 6 below.

[Table 6]

During the culture period of mesenchymal stem cell spheroids (MiB), the 5 mL tubes and parafilms to be 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 weighed again. If a weight difference of 0.2 μg or more from the previous weight was confirmed, the zero point was reset and the weight was measured again.

After 48 hours of culture, mesenchymal stem cell spheroids (MiB) cultured at 0, 4,800, and 9,600 in 5 mL tubes were collected in 3 replicates, and then suspended in 200 μL of DPBS. The inlet of the 5 mL tube was sealed with parafilm, a hole was poked with a 30 gauge needle, and dried for 2 days using a freeze dryer. The weight of the freeze-dried sample was measured three times using an electronic balance, and the average of the measurements was calculated to calculate the weight. From the weight of the freeze-dried sample, 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. The calculated dry weight was divided by the number of each mesenchymal stem cell spheroid (MiB) to obtain the weight of 1 mesenchymal stem cell spheroid (MiB).

② Results of dry weight measurement of mesenchymal stem cell spheroids (MiB ) and mesenchymal stem cell spheroids (MiB)

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 7 below. The single mesenchymal stem cell spheroid (MiB) dry weight measurement deviation between the two experimenters was shown in Fig. 25, and the linear trend result for the mesenchymal stem cell spheroid (MiB) weight is shown in Fig. 26.

[Table 7]

Referring to Table 7 above and Figure 25, 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 ±S.D.), respectively, and there was no statistically significant difference.

In addition, the dry weight data of 0, 4,800, and 9,600 mesenchymal stem cell spheroids from two experimenters were integrated and used to obtain a linear trend line, and the experiment was found to be statistically significant with ^R2 =0.9917 (Fig. 26).

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 7.

Example 5. Proteomic analysis of mesenchymal stem cell spheroids (MiB) of the present invention

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

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

For the analysis, the sample was placed in a 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 analysis (BCA) and separated on an SDS-PAGE gel to perform in-gel digestion. The gel was fractionated by molecular weight according to the method of Lee, Sang-Yeop, et al., and the gel was washed with a solution prepared with 30% methanol, 10 mM ammonium bicarbonate, and 50% acetonitrile. After washing, the gel was dried and reduced with 10 mM dithiothreitol and 55 mM iodoacetamide, followed by tryptic digestion 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.

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 minutes 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).

Protein identification was based on monoisotopic mass selection, precursor mass tolerance ±5 Da, fragment mass tolerance ±0.8 Da, two miscleavages, and a corrected carbamidomethylcysteine. Individual spectra were accepted based on a Mascot ion threshold score of 0.05 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.

Single cells and mesenchymal stem cell spheroids (MiB) of the present invention were tested with three samples per group, and the results are as shown in Figure 27.

As can be confirmed in Figure 27, 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%.

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

In order to confirm the correlation and protein composition differences between groups using the proteins that constitute 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 cell and mesenchymal stem cell spheroids were located oppositely, indicating that the types of the composed proteins were clearly different, and the results are as shown in Figure 28. In addition, in the heatmap analysis showing the correlation between groups in rows, columns, and colors, the differences in protein composition and content between single cell and mesenchymal stem cell spheroids were confirmed, and the results are as shown in Figure 29.

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

The increase/decrease distribution of proteins in the mesenchymal stem cell spheroids (MiB) of the present invention compared to single cells was analyzed. The increase/decrease distribution of proteins is shown in Fig. 30, and the results of confirming the increased structural proteins in the mesenchymal stem cell spheroids (MiB) of the present invention compared to single cells are as shown in Table 8.

[Table 8]

As a result of analysis of Volcano plots that visually represent statistical significance for changes in protein composition (Fig. 30), the protein that showed the greatest change in the mesenchymal stem cell spheroids (MiB) of the present invention compared to single cells was found to be Tenascin (TNC). Tenascin is a protein expressed when tissues are damaged or inflamed, and is known to be involved in cell proliferation response and promote healing through activation of the epidermal growth factor receptor (EGFR) and inflammatory signaling pathways.

Likewise, Fibronectin (FN1) is one of the proteins whose levels were found 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.

As such, the proteins increased in the mesenchymal stem cell spheroids (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 the structure increased, and it is presumed that this change is due to the induction of self-assembly, which is the production principle of the mesenchymal stem cell spheroids (MiB) of the present invention.

In addition, proteoglycan-4 (PRG4) was shown to be a protein specifically expressed only in mesenchymal stem cell spheroids (MiB). PRG4 is known to help wound healing by reducing fibrosis and enhancing angiogenesis by inducing adipocytes to the wound site by regulating molecular pathways related to wound healing.

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

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 (31)

(a) a step of producing a spheroid-shaped cell aggregate by culturing mesenchymal stem cells in three dimensions; and (b) a step of separating mesenchymal stem cell spheroids having an average diameter of 10 μm to 300 μm from the spheroid-shaped cell aggregate; A method for producing a mesenchymal stem cell spheroid comprising: A method for producing a mesenchymal stem cell spheroid in claim 1, wherein in step (a), the mesenchymal stem cell is 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. A method for producing a mesenchymal stem cell spheroid, further comprising, before step (a), a step of isolating mesenchymal stem cells (MSCs) and culturing them in two dimensions. A method for producing a mesenchymal stem cell spheroid, wherein, in claim 1, before step (a), the method further comprises a step of treating mesenchymal stem cells with at least one protein-decomposing enzyme selected from the group consisting of trypsin, collagenase, papain, and elastase. A method for producing a mesenchymal stem cell spheroid, wherein in step (a), mesenchymal stem cells self-assemble to form a spherical spheroid-shaped cell aggregate. In the first paragraph, step (a) is a method for producing a mesenchymal stem cell spheroid, wherein 50 to 5,000 mesenchymal stem cells are treated per well to culture the mesenchymal stem cells in three dimensions to produce a spherical spheroid-shaped cell aggregate. In claim 1, step (a) is a method for producing a mesenchymal stem cell spheroid, wherein the step comprises centrifuging mesenchymal stem cells to form cell aggregates. In claim 7, step (a) is a method for producing a mesenchymal stem cell spheroid, wherein 50 to 5,000 mesenchymal stem cells are centrifuged at 100 to 1,000 xg to produce a spherical spheroid-shaped cell aggregate. A method for producing mesenchymal stem cell spheroids, wherein in step (b), cell aggregates having an average diameter of 10 μm to 300 μm are separated to obtain mesenchymal stem cell spheroids having a uniform diameter. A method for producing mesenchymal stem cell spheroids, wherein in claim 1, step (b) comprises a step of passing the spheroid-shaped cell aggregate produced in step (a) through a cell strainer having a diameter of 300 μm or less. A method for producing mesenchymal stem cell spheroids in claim 1, wherein the average diameter of the mesenchymal stem cell spheroids in step (b) is 10 μm to 300 μm. A method for producing a mesenchymal stem cell spheroid in claim 1, wherein the wet weight of the mesenchymal stem cell spheroid is 1 to 6 μg. A method for producing a mesenchymal stem cell spheroid in claim 1, wherein the dry weight of the mesenchymal stem cell spheroid is 250 to 500 ng. A method for producing a mesenchymal stem cell spheroid in claim 1, wherein in step (b), the mesenchymal stem cell spheroid comprises mesenchymal stem cells (MSCs) and an extracellular matrix (ECM). A method for producing a mesenchymal stem cell spheroid in claim 14, wherein the extracellular matrix comprises at least one selected from the group consisting of collagen, fibronectin (FN1), hyaluronic acid (HA), elastin (EL), and glycosaminoglycan (GAG). A method for producing a mesenchymal stem cell spheroid in 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). A method for producing a mesenchymal stem cell spheroid in claim 1, wherein the mesenchymal stem cells included in the mesenchymal stem cell spheroid include 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. A method for producing a mesenchymal stem cell spheroid in claim 1, wherein the mesenchymal stem cells included in the mesenchymal stem cell spheroid include 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. A method for producing a mesenchymal stem cell spheroid in claim 1, wherein the mesenchymal stem cells included in the mesenchymal stem cell spheroid include 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. In claim 1, the mesenchymal stem cell spheroid is a method for producing a mesenchymal stem cell spheroid, 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). A mesenchymal stem cell spheroid having a uniform diameter manufactured according to Article 1, Contains mesenchymal stem cells (MSCs) and extracellular matrix (ECM). Mesenchymal stem cell spheroids with an average diameter of 10 μm to 300 μm. A composition comprising mesenchymal stem cell spheroids having a uniform diameter manufactured according to claim 1 and hyaluronic acid. A composition according to claim 22, wherein the composition comprises mesenchymal stem cell spheroids at a concentration of 1 to 2 × 10 ⁵ cells/mL. A composition according to claim 22, wherein the composition comprises hyaluronic acid at a concentration of 2 to 50 mg/mL. A composition in claim 22, wherein the hyaluronic acid comprises hyaluronic acid, a hyaluronic acid salt or a mixture thereof. A composition in claim 22, wherein the weight average molecular weight of the hyaluronic acid is 100 Da to 10,000 kDa. A pharmaceutical composition for skin regeneration comprising a mesenchymal stem cell spheroid of claim 21. A pharmaceutical composition for treating skin wounds or scars, comprising mesenchymal stem cell spheroids of claim 21. A pharmaceutical composition according to claim 28, 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 scleroderma, a scar caused by a wound, a scar caused by a diabetic foot ulcer, a keloid scar, an atrophic scar, and a stretch mark. A pharmaceutical composition in claim 28, 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. A pharmaceutical composition according to claim 28, 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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