Method for producing stem cell-derived exosomes by cell elongation and its applications

Abstract

This invention confirms that when a hydrogel in a sol state, composed of a polymer capable of sol-gel phase transition, is mixed with stem cells and the phase transition is induced to a gel state, stem cells can be cultured uniformly and at high density in a 3D state. Furthermore, we confirmed that culturing stem cells using the above method allows for the stretching of stem cells through the regulation of the hydrogel structure, thereby transmitting physical stimuli to the cells, activating intracellular integrin-FAK and catenin-Wnt signaling, increasing exosome production efficiency, and increasing the amount of functional components. Furthermore, we confirmed that exosomes produced by the present invention generate miRNA components that can regulate angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced by the present invention can be usefully utilized as compositions for angiogenesis, tissue regeneration, or anti-inflammatory purposes.

Description

Detailed description of the invention

[0001] [Technical field] The present invention relates to a method for producing functional exosomes derived from stem cells with increased cell elongation and its applications. More specifically, the present invention relates to a method for increasing stem cell-derived exosome production by transmitting physical stimuli through cell stretching and its applications, comprising the steps of: mixing a hydrogel in a sol state containing polymer chains capable of sol-gel phase transition having ligands that can bind to cellular integrins and capable of sol-gel phase transition with stem cells; and transitioning the hydrogel to a gel state to produce a 3D hydrogel in which stem cells are uniformly captured in three dimensions, and culturing the stem cells.

[0002] [Background technology] Stem cells possess anchorage-dependent properties, meaning they attach to and survive in a culture vessel, and grow there. Stem cell exosomes are produced by stem cells and secreted into the culture medium. These exosomes can then be separated, concentrated, and purified to be manufactured as pharmaceuticals.

[0003] To produce stem cell exosomes, a supply of cell adhesion substrate and culture medium that allows stem cells to adhere, survive, and maintain function is essential. Industrial-scale production of stem cell exosomes in a typical monolayer culture (2-dimensional culture conditions; 2D) environment requires a large number of culture vessels and large volumes of culture medium, resulting in low efficiency. To overcome the low efficiency of a 2D environment with limited substrate area for cell adhesion, a technology was needed to produce exosomes through high-density culture in a 3D environment that could provide a substrate on a small surface area to which many cells could adhere. When anchorage-dependent stem cells are cultured in suspension culture without a cell adhesion substrate, the anoikis phenomenon occurs, leading to cell damage and death. 3D cell adhesion substrates are required for high-density culture that increases the volume of tissue that can adhere to cells and suppresses cell death. To achieve this objective, porous supports, hydrogels, and microspheroid carriers are being utilized as 3D cell adhesion substrates.

[0004] In the case of a porous scaffold, most cells attach and grow along the surface of the support, forming a membrane that prevents the exchange of oxygen, nutrients, and waste products into the support, resulting in necrosis in the center. In the case of a microsphere support, although the surface area on which cells can attach theoretically increases compared to a 2D environment, aggregation occurs due to binding between microspheres during suspension culture, causing cells on the surface of the microspheres to detach, making long-term culture difficult.

[0005] Phase-transition hydrogels are defined as hydrogels that are easily mixed with cells in their sol state before crosslinking, and that can transition to a gel state after crosslinking. The hydrogel polymer solution in its pre-crosslinking solution state can be uniformly mixed with cells, and after crosslinking, cells can be uniformly distributed in 3D within the hydrogel. Compared to porous supports and microsphere supports, they have the advantage of good interpenetrating network (IPN) connectivity between polymer chains, allowing for smooth transport of oxygen, nutrients, and metabolites.

[0006] However, hydrogels have fragile biochemical properties that make them susceptible to degradation by matrix metalloproteinases (MMPs) secreted by cells. If their physical properties are too rigid, cell binding and elongation are inhibited, resulting in a decline in the molecular biological function of cells. The interaction between cells and polymer chains is greatly influenced by the polymer components that make up the hydrogel. Furthermore, the structural and physical properties of the hydrogel change depending on the concentration, molecular weight, and degree of crosslinking of the polymers that make up the hydrogel. Consequently, cell growth, migration, activity, elongation, and gene expression are also altered by the structure and physical properties of the hydrogel, resulting in changes in the biological function and activity of cells, and significantly affecting the exosome generation rate and the gene composition within exosomes.

[0007] There is a need for technology that can generate stem cell exosomes and exosomes with enhanced biological function through research that optimizes the interaction between polymer chains and cells within 3D hydrogels by adjusting the structure and physical properties of 3D hydrogels to act as cell adhesion substrates that enable high-density cell culture in 3D culture environments.

[0008] [Overview of the prefecture] [Problems the invention aims to solve] Therefore, in this invention, we have made diligent efforts to efficiently generate stem cell-derived exosomes by adjusting the structure and physical properties of the hydrogel. As a result, we have confirmed that by mixing stem cells with a polymer solution in a sol state that is capable of sol-gel phase transition and contains polymer chains having ligands that can bind to cellular integrins, and then transitioning to a gel state after a cross-linking reaction, it is possible to culture stem cells uniformly and at high density in 3D within a 3D hydrogel. By providing stem cells with a 3D cell adhesion substrate using a hydrogel, it is possible to provide an environment in which cells can be cultured at a density 10 to 100 times higher per unit area compared to a 2D culture environment, allowing for the cultivation of a large number of cells even in a small culture vessel, and improving the efficiency of exosome generation.

[0009] Furthermore, culturing stem cells in a 3D hydrogel provides a method to control the interaction between stem cells and the hydrogel, thereby regulating the gene expression and function of cells by adjusting the physicochemical properties and structural properties, including pore size, of the hydrogel. Depending on the properties of the hydrogel, the physiological activity, function, gene expression, exosome generation rate, and exosome composition of cells are altered.

[0010] This invention provides a method for stretching cells by adjusting the physicochemical and structural properties of a hydrogel, inducing activation of the integrin-FAK signaling pathway in accordance with the degree of cell stretching, and thereby promoting the exosome production rate of cells. Furthermore, it provides a method for maximizing cell-cell junctions by adjusting the hydrogel structure, activating the β-catenin-Wnt signaling pathway to activate intracellular gene expression, and thereby controlling the miRNA composition within exosomes produced by cells.

[0011] The present invention aims to provide an industrial-grade culture base for exosome production through high-density culture of stem cells in a 3D hydrogel, and to provide a method for producing a composition that can increase the exosome production rate through cell elongation and improve anti-inflammatory, tissue regeneration, and angiogenesis properties within the exosomes.

[0012] [Means for solving the problem] To achieve the above objectives, the present invention provides (a) a step of mixing a hydrogel in a sol state, which comprises / includes a polymer chain having a ligand capable of binding to cellular integrins and capable of undergoing a sol-gel phase transition, with stem cells; and (b) The hydrogel is subjected to a phase transition to a gel state to produce a 3D hydrogel in which stem cells are uniformly collected in three dimensions, and the stem cells are cultured; the present invention provides a method for increasing stem cell-derived exosome production by transmitting physical stimuli through cell stretching.

[0013] In a preferred embodiment of the present invention, the above method allows the ligand of the hydrogel to directly bind to the cell and stretch the cell without the need for physical equipment.

[0014] In another preferred embodiment of the present invention, the hydrogel having a ligand capable of binding to the integrin may be a natural polymer selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin, or a hydrogel obtained by copolymerizing the natural polymer with a synthetic polymer selected from the group consisting of PEG (Polyethylene Glycol), PLA (Polylactic Acid), and PGA (Polyglycolic Acid).

[0015] In another preferred embodiment of the present invention, in the above step (b), a crosslinking agent can be added to the mixture in step (a) to cause a phase transition of the hydrogel into a gel state.

[0016] In another preferred embodiment of the present invention, in order to enhance the physicochemical properties of the 3D hydrogel in the above step (b), in the above step (a), one or more polymer substances selected from the group consisting of PEG, PLGA, PGA, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid can be additionally mixed. By enhancing the above properties, "gel compaction" caused by the detachment of the hydrogel from the culture vessel due to the contraction force of cells can be prevented.

[0017] In another preferred embodiment of the present invention, in order to suppress the degradation of the 3D hydrogel by cells in the above step (b) and enhance its stability, in the above step (a), one or more matrix metalloproteinase inhibitors (MMPs) selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9 can be inhibited, and one or more MMP inhibitors selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat can be further mixed.

[0018] In another preferred embodiment of the present invention, in order to suppress the degradation of the 3D hydrogel by cells in the above step (b) and enhance its stability, in the above step (a), one or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid can be further mixed.

[0019] In another preferred embodiment of the present invention, the 3D hydrogel in the above step (b) can have a porous structure with a size of 50 nm to 300 nm, provide cell-cell binding, and have structural properties that promote cell stretching.

[0020] In another preferred embodiment of the present invention, the stem cells cultured in the above step (b) have a form that extends in a three-dimensional axial plane, and the cells can extend to a length of 15 μm to 400 μm.

[0021] In another preferred embodiment of the present invention, the above method can impregnate and culture stem cells at a high density of 1.0×10 4 ~5.0×10 6 per mL of hydrogel. In another preferred embodiment of the present invention, in the above step (b), the stem cells cultured through the regulation of the physicochemical properties and structural properties of the hydrogel can activate the integrin-FAK-JNK-β-catenin signal transduction pathway.

[0022] In another preferred embodiment of the present invention, the stem cells cultured through the regulation of the hydrogel physical properties and structural properties in the above step (b) can activate the Wnt signal transduction pathway and increase the expression of Wnt signal transduction sub-target genes.

[0023] In another preferred embodiment of the present invention, the exosomes derived from stem cells produced by the above method can increase the composition of any one or more angiogenesis-regulating miRNAs selected from the group consisting of let-7b-3b and miR126, or any one or more inflammation-regulating miRNAs selected from the group consisting of miR145-5p, miR146a-5p and miR185-5p.

[0024] In another preferred embodiment of the present invention, the stem cells may be adult stem cells or mesenchymal stem cells, and preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, muscle-derived stem cells, cardiomyocyte-derived stem cells, peripheral nerve-derived stem cells, synovial membrane-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

[0025] To achieve other objectives, the present invention provides a composition for angiogenesis, tissue regeneration, or anti-inflammatory purposes, comprising stem cell-derived exosomes produced by the above method as an active ingredient. In one preferred embodiment of the present invention, the above composition may be a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

[0026] [Effects of the invention] This invention confirms that when a hydrogel in a sol state, composed of a polymer capable of sol-gel phase transition, is mixed with stem cells and the phase transition is induced to a gel state, stem cells can be cultured uniformly and at high density in a 3D state. Furthermore, it was confirmed that culturing stem cells using the above method allows for the stretching of stem cells through the regulation of the hydrogel structure, thereby transmitting physical stimuli to the cells, activating intracellular integrin-FAK and catenin-Wnt signaling, increasing exosome production efficiency, and increasing the amount of functional components. It was confirmed that exosomes produced by the method of this invention have increased levels of miRNA components that can regulate angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced by the method of this invention can be usefully utilized as compositions for angiogenesis, tissue regeneration, or anti-inflammatory purposes. [Brief explanation of the drawing]

[0027] [Figure 1]Figure 1 shows images of cell characteristics observed in a 2D culture environment for anchorage-dependent stem cells. AT-MSC: Adipose-derived mesenchymal stem cells; BM-MSC: Bone marrow-derived mesenchymal stem cells; CB-MSC: Umbilical cord blood-derived mesenchymal stem cells; MyoCSC: Cardiomyocyte-derived stem cells; PNSC: Peripheral nerve-derived stem cells; SM-MSC: Muscle-derived stem cells; Syn-MSC: Synovial membrane-derived mesenchymal stem cells; UC-MSC: Umbilical cord-derived mesenchymal stem cells. [Figure 2] Figure 2 is a flow cytometry histogram image showing the immune expression characteristics of cardiomyocyte-derived stem cells. [Figure 3] Figure 3 shows images observing the differentiation characteristics of anchorage-dependent stem cells into adipocytes and osteoblasts. AT-MSC: Adipose-derived mesenchymal stem cells; BM-MSC: Bone marrow-derived mesenchymal stem cells; MyoCSC: Cardiomyocyte-derived stem cells; PNSC: Peripheral nerve-derived stem cells. [Figure 4] Figure 4 shows images and results of (A) the porous structure of the hydrogel and (B) the pore size at different fibrinogen concentrations. [Figure 5] Figure 5 shows data measuring (A) the degree of microfilament formation, which represents the degree of cell elongation, and (B) the microfilament area per cell within the fibrin hydrogel, based on fibrinogen concentration. [Figure 6] Figure 6 shows images of integrin-β1 and p-FAK expression in cardiomyocyte-derived stem cells within a fibrin hydrogel, depending on fibrinogen concentration. [Figure 7] Figure 7 shows (A) images of p-FAK, p-JNK, and p-β-catenin expression levels measured via Western blot in cardiomyocyte-derived stem cells in a 3D fibrin hydrogel based on fibrinogen concentration, and (B) measured values ​​of p-FAK, p-JNK, and p-β-catenin expression levels corrected for the expression levels of cardiomyocyte-derived stem cells in a 2D culture environment. *, p<0.05 relative to the 2D culture environment expression level; **, p<0.01 relative to the 2D culture environment expression level. [Figure 8]Figure 8 shows the results of Wnt signaling mRNA expression in cardiomyocyte-derived stem cells in a 3D fibrin hydrogel, corrected for mRNA expression rates of cardiomyocyte-derived stem cells in a 2D culture environment. (A) Expression rates of Wnt ligand-related mRNAs, (B) Expression rates of Wnt receptor-related mRNAs, and (C) Expression rates of sub-mRNAs targeted by the Wnt signaling pathway. *, p<0.05 compared to the 2D culture environment; **, p<0.01 compared to the 2D culture environment. [Figure 9] Figure 9 shows a graph of (A) immunofluorescence staining to observe the expression of exosomes of cardiomyocyte-derived stem cells in a 3D fibrin hydrogel according to fibrinogen concentration, and (B) the immunofluorescence intensity quantified. **p<0.01 for fibrin hydrogels prepared with 5 mg and 10 mg fibrinogen. [Figure 10] Figure 10 shows data obtained by measuring (A) the number of exosomes per mL measured using a nanoparticle tracking analyzer and (B) the protein content, in order to determine the exosome generation rate of cardiomyocyte-derived stem cells in 3D fibrin hydrogel and 2D culture environments based on fibrinogen concentration. *, p<0.01 compared to the 2D culture environment. [Figure 11] Figure 11 shows the diameter of exosomes from cardiomyocyte-derived stem cells in 3D fibrin hydrogel and 2D culture environments, measured using a nanoparticle tracking analyzer, based on fibrinogen concentration. The figures represent (A) the average diameter and (B) the modal diameter of the exosomes. [Figure 12] Figure 12 is a flow cytometer dot plot image used to analyze the immunoexpression characteristics of cardiomyocyte-derived stem cell exosomes generated in a 3D fibrin hydrogel and a 2D culture environment. [Figure 13]Figure 13 shows the measured values ​​of exosome generation rates by stem cell sources in 3D fibrin hydrogel and 2D culture environments. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; CB-MSC, umbilical cord blood-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; Syn-MSC, vital membrane-derived mesenchymal stem cells; UC-MSC, umbilical cord-derived mesenchymal stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Figure 14] Figure 14 shows the measured exosome generation rate based on the seeding density of cardiomyocyte-derived stem cells in a 3D fibrin hydrogel. *, p<0.05 relative to 1.0E+04; **, p<0.01 relative to 1.0E+04. [Figure 15] Figure 15 shows the (A) microfiber formation and (B) exosome generation rate of cardiomyocyte-derived stem cells in a 3D culture environment using hydrogel-based natural polymer components. *, p<0.05 for gelatin, hyaluronic acid, and chondroitin; **, p<0.01 for gelatin, hyaluronic acid, and chondroitin. [Figure 16] Figure 16 shows the measured values ​​of (A) microfiber formation and (B) exosome generation rate of cardiomyocyte-derived stem cells in synthetic polymer hydrogels and synthetic polymer-fibrin mixed hydrogels. *, p<0.05 for synthetic polymer hydrogel; **, p<0.01 for synthetic polymer hydrogel. [Figure 17] Figure 17 shows the miRNA expression characteristics in cardiomyocyte-derived stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments. The miRNA expression rates are expressed as ratios corrected for bone marrow-derived mesenchymal stem cells (BM-MSCs). * indicates p<0.05 relative to the 2D culture environment; ** indicates p<0.01 relative to the 2D culture environment. [Figure 18]Figure 18 is a graph comparing the fibroblast growth-inducing ability of stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments. The comparison was performed using a ratio corrected for the cell number of the group to which exosome-free culture medium (vehicle) was added. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Figure 19] Figure 19 is a graph comparing the vascular endothelial cell growth-inducing ability of stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments. The comparison was performed using a ratio corrected for the cell number of the group to which exosome-free culture medium (vehicle) was added. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Figure 20] Figure 20 shows the results of measuring the ability of stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments to induce vascular endothelial cell growth. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Figure 21] Figure 21 shows the results of measuring the ability of stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments to suppress inflammatory cell TNF-α secretion. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Figure 22]Figure 22 shows the measured ability of stem cell exosomes generated in 3D fibrin hydrogel and 2D culture environments to suppress inflammatory cell IL-1β secretion. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiomyocyte-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p<0.05 compared to 2D culture environment; **, p<0.01 compared to 2D culture environment. [Modes for carrying out the invention]

[0028] The present invention will be described in detail below. From one perspective, the present invention provides for the steps of (a) mixing a hydrogel in a sol state with stem cells, comprising a polymer chain having a ligand capable of binding to cellular integrins and capable of undergoing a sol-gel phase transition; and (b) A method for increasing stem cell-derived exosome production by transmitting physical stimuli through cell stretching, comprising the step of inducing a phase transition of the above hydrogel to a gel state to produce a 3D hydrogel in which stem cells are uniformly captured in three dimensions, and culturing the stem cells.

[0029] More specifically, the present invention relates to (a) the step of mixing a hydrogel in a sol state and stem cells, which comprises a polymer chain having ligands capable of binding to integrins of cells and capable of undergoing a sol-gel phase transition; (b) The above hydrogel is subjected to a phase transition to a gel state to produce a 3D hydrogel in which stem cells are uniformly collected in three dimensions, and the stem cells are cultured in this step; (c) A step of stretching the cell through 3D binding between the cell and polymer chain, transmitting physical stimuli into the cell to enhance the integrin-FAK signaling pathway, and increasing the rate of stem cell-derived exosome production; and, (d) A method for enhancing the β-catenin-Wnt signaling pathway through cell-cell junctions and increasing miRNA components in stem cell-derived exosomes that have anti-inflammatory, tissue regeneration, and angiogenesis effects.

[0030] In the present invention, the stem cells may be adult stem cells or mesenchymal stem cells, and preferably, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, cardiomyocyte-derived stem cells, muscle-derived stem cells, peripheral nerve-derived adult stem cells, synovial membrane-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

[0031] In the present invention, the above method allows the ligand of the hydrogel to bind to cells and stretch the cells without the need for physical equipment. In the present invention, the hydrogel having a ligand capable of binding to the above-mentioned integrin is a natural polymer selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin, or The hydrogel can be copolymerized with the above-mentioned natural polymeric substance and a synthetic polymeric substance selected from the group consisting of PEG (Polyethylene Glycol), PLA (Polylactic Acid), and PGA (Polyglycolic Acid), and is preferably fibrinogen, collagen, or gelatin.

[0032] In the present invention, step (b) above involves adding a crosslinking agent to the mixture from step (a) to cause the hydrogel to undergo a phase transition to a gel state.

[0033] The above polymer possesses a ligand (native ligand) that can directly bind to cellular integrins, and is therefore applicable to the present invention. Among these polymers, collagen, fibrin, and gelatin, for example, allow for adjustment of their polymer content and cross-linking density, enabling the modification of the microstructure and physical properties within the hydrogel. As a result, the rate and composition of exosome production in cells can be controlled.

[0034] On the other hand, the above-mentioned hydrogels have biodegradable properties, meaning they are easily degraded by matrix metalloproteinases (MMPs) secreted by cells. Furthermore, a drawback has emerged: after the polymer chains bind to cells, the hydrogel shrinks due to cell-mediated contraction, resulting in the loss of its structural function as a 3D substrate. To overcome these biochemical and physical drawbacks, technologies to enhance biodegradability and structural properties are required. Stability against biodegradation can be increased through the introduction of MMP inhibitors or anti-fibrinolytic agents. Additionally, hydrogels with enhanced physical properties can be utilized through copolymerization with polymers that are not degraded by MMPs and have high physical strength, such as PEG, PLGA, PGF, chitosan, and hyaluronic acid, thereby increasing resistance to cell-mediated contraction.

[0035] Specifically, in order to adjust the physical strength of the 3D hydrogel in step (b) above, one or more polymer materials selected from the group consisting of PEG, PLGA, PGF, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid may be further mixed into the mixture in step (a) above.

[0036] Furthermore, in order to inhibit cellular degradation of the 3D hydrogel in step (b) above and enhance its stability, step (a) above may include one or more matrix metalloproteinase inhibitors selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat, which can inhibit any one or more matrix metalloproteinases selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9; or One or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid may be further added.

[0037] In the present invention, the 3D hydrogel in step (b) above has a porous structure of 50 nm to 300 nm size and can have structural properties that support cell-cell bonding and simultaneously promote cell stretching.

[0038] The stem cells cultured in step (b) above exhibit an elongated shape in the 3D axial plane, and the cells can elongate to lengths of 15 μm to 400 μm. Furthermore, the above method yields 1.0 × 10 per mL of hydrogel. 4 ~5.0×10 6 Stem cells can be cultured by impregnating them with high density.

[0039] Since hydrogels can provide a 3D cell attachment matrix to which cells can adhere at high density, the present invention can provide a method to increase exosome production efficiency by culturing at a higher density than normal 2D culture through high-density culture. In the case of lipid-dependent cells such as mesenchymal stem cells, in a 2D environment, cm 2 5.0 x 104 In contrast, in the case of a 3D environment, it is impossible to culture cells above a certain cell density. For example, per cm 2 it is possible to culture 0.5 to 5.0×10 6 or more cells, and a cell adhesion substrate capable of culturing cells at a density 10 to 100 times or more higher than that in 2D can be provided.

[0040] The production rate of exosomes is directly proportional to the number of cells that produce exosomes. In particular, in the case of stem cells, which are anchorage-dependent cells, the area of the substrate to which they can adhere is an absolute variable that determines the number of cells that can be cultured, and the exosome production yield is determined by the number of cells to be cultured. The present invention provides a 3D substrate using a hydrogel to which cells can adhere at high density, and provides a technology capable of generating a large-scale level of exosomes even in a small-sized culture vessel. In particular, by using a sol-gel phase transition hydrogel, there is an advantage that it can be mixed with cells in a sol state and phase-converted into a gel state to uniformly impregnate stem cells into the 3D hydrogel. When culturing stem cells in a T175 flask, the maximum number of stem cells that can be cultured on the area of the flask, which is 175 cm 2 is 7 to 8E+06, but the number of cells that can be cultured in a 1-cm-thick 3D hydrogel can be up to 8E+08.

[0041] Specifically, the present invention can regulate the exosome production rate of stem cells and their ability to induce inflammation, tissue regeneration, and angiogenesis by modifying the structure and physicochemical properties of hydrogels. It provides a method for inducing cell stretching by having polymer chains constituting nanostructures within the hydrogel bind to cells, thereby activating integrin, FAK, β-catenin, and Wnt signaling pathways in response to physical stimuli compared to a 2D environment. The invention provides a method for regulating cellular function by modifying the structure and physicochemical properties of hydrogels, ultimately increasing the efficiency of stem cell exosome production and enhancing functional components. Coarse nanostructures within the hydrogel increase stem cell stretching and intercellular junctions, while stem cells within dense nanostructures decrease these. The degree of cell stretching and intercellular junctions alters the activity of FAK, integrin, β-catenin, and Wnt signaling pathways, consequently affecting the exosome production rate within stem cells and the composition of functional components within exosomes.

[0042] In one specific embodiment of the present invention, a hydrogel can be produced using fibrinogen, and more specifically, stem cell-derived exosomes can be produced by a method comprising the steps of (1) mixing a hydrogel polymer solution in a sol state with stem cells; and (2) adding a crosslinking agent to the mixture to produce a hydrogel in a gel state in which stem cells are uniformly captured in 3D, and culturing the stem cells.

[0043] Furthermore, the structural properties and physicochemical properties of hydrogels can be adjusted by the content of the constituent polymers and the degree of cross-linking reactions. A higher polymer content results in a more faithful structure, while a lower content allows for the production of a coarser structure. However, the drawbacks of coarse hydrogels can be compensated for by adding MMP inhibitors, anti-fibrinolytic agents, and synthetic polymers, which enhance the stability against degradation of coarse hydrogels and compensate for the fragile physical strength that causes the hydrogel to compact due to cell contraction and detach from the culture vessel.

[0044] In another specific embodiment of the present invention, it was confirmed that the structural properties and physical characteristics of the fibrin hydrogel can be adjusted by controlling the fibrinogen concentration. Higher fibrinogen concentrations reduce pore size and narrow pore spacing, allowing for the production of a dense and faithful structure, while lower fibrinogen content allows for the production of a hydrogel with a coarser structure.

[0045] In other words, in this invention, when a hydrogel is produced with fibrinogen at a concentration of 1.25 mg / mL to 5 mg / mL, the 3D hydrogel has a porous structure with a size of 50 nm to 300 nm, preferably 100 nm to 300 nm, and it was confirmed that the stem cells in the fibrin hydrogel composed of low-concentration fibrinogen extend to a length of 15 μm to 400 μm, and that cell-cell junctions increase (Figures 4 and 5).

[0046] Furthermore, when stem cells were cultured using the fibrin hydrogel produced according to the present invention, it was confirmed that the FAK, integrin, Wnt, and β-catenin cell signaling pathways were activated in the coarse-structured fibrin hydrogel, that is, in proportion to the degree of stem cell elongation and intercellular junction (Figures 6-9), and that exosome production increased (Figures 10 and 11).

[0047] Furthermore, a comparison of exosomes produced in the 3D hydrogel culture environment of the present invention with those produced in 2D culture revealed no significant differences in exosome size or immune expression characteristics (Figure 12). However, a significant increase was observed in exosome generation by stem cell sources (Figure 13), angiogenesis, tissue regeneration, and anti-inflammatory regulatory miRNA expression (Figure 17). In addition, exosomes produced in the 3D hydrogel culture environment of the present invention showed a remarkable increase in angiogenesis, tissue regeneration, and anti-inflammatory regulatory effects compared to exosomes produced in 2D culture (Figures 18-22).

[0048] In this invention, the degree of elongation of impregnated stem cells can be regulated by modifying the nanostructure of the hydrogel. It was confirmed that as cell elongation increases, physical stimulation activates the cell signaling pathways of stem cells, leading to increased exosome production. Furthermore, it was confirmed that exosomes produced by the method of this invention showed an increase in the types of cell growth, wound repair, angiogenesis, inflammation-modulating proteins, and miRNAs within the exosomes, thereby improving the angiogenesis, tissue regeneration, and anti-inflammatory efficacy of the exosomes.

[0049] In another aspect, the present invention relates to a composition for angiogenesis, tissue regeneration, or anti-inflammatory purposes, comprising stem cell-derived exosomes produced by the above method as an active ingredient. In the present invention, the above composition may be a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

[0050] The above-mentioned angiogenic composition can induce or increase the migration of vascular endothelial cells and can promote angiogenesis in diseases requiring angiogenesis. The diseases requiring the above-mentioned angiogenesis are one or more selected from the group consisting of wounds, burns, ulcers, necrosis, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease, and alopecia.

[0051] The above tissue regeneration composition can be used to regenerate tissues selected from skin, cartilage, bone, blood vessels, brain, liver, heart, ligaments, muscles, spinal cord, blood, bone marrow, lungs, teeth, nerves, cornea, retina, esophagus, spine, elongation, pancreas, or urethra, but is not limited to these. Furthermore, the above tissue regeneration composition can be used to improve skin condition, fill in wrinkles, or shape the contours of the face or body. In addition, it can be usefully used as a dermal filler.

[0052] The above-mentioned anti-inflammatory composition is a pharmaceutical composition for the prevention or treatment of an inflammatory disease, wherein the inflammatory disease is selected from the group consisting of multiple sclerosis, ischemic stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, or amyotrophic lateral sclerosis.

[0053] In the present invention, the above-mentioned inflammatory disease is any one selected from the group consisting of atopic dermatitis, psoriasis, dermatitis, allergy, arthritis, rhinitis, otitis media, pharyngitis, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, inflammatory bowel disease, ankylosing spondylitis, systemic lupus erythematosus (SLE), atherosclerosis, asthma, arteriosclerosis, edema, rheumatoid arthritis, delayed allergy (type IV allergy), graft rejection, graft-versus-host disease, autoimmune encephalomyelitis, arthritis, cystic fibrosis, diabetic retinopathy, rhinitis, ischemic-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy.

[0054] The pharmaceutical compositions of the present invention can be formulated and used in various forms according to conventional methods. For example, they can be formulated into oral dosage forms such as acids, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated and used in the form of topical preparations, suppositories, and sterile injections. Depending on the formulation, pharmaceutically acceptable carriers, excipients, and diluents may be further included. Furthermore, they can be formulated and used in the form of topical preparations such as acids, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and sterile injections, according to conventional methods.

[0055] Examples of the carriers, excipients, and diluents mentioned above include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. When formulating or preparing the above pharmaceutical composition, it is compounded using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients.

[0056] Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. Such solid preparations are prepared by mixing the above-mentioned composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as humectants, sweeteners, flavorings, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suitable suppository bases include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerogenatin.

[0057] As used in this invention, the term "administration" means providing the pharmaceutical composition of the present invention to an individual in any suitable manner. The pharmaceutical composition of the present invention can be administered in a therapeutically effective dose, which is the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human, as researchers, veterinarians, physicians, or other clinicians might consider, i.e., the amount that induces relief of the symptoms of the disease or disorder being treated. It will be obvious to those skilled in the art that the therapeutically effective dose and number of administrations of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art and may be adjusted according to various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients in the composition, the type of formulation, the patient's age, weight, general health, sex and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and drugs used concurrently. The pharmaceutical composition of the present invention can be administered to an individual by various routes. For example, it can be administered intravenously, intraperitoneally, intramuscularly, intra-arterially, orally, intracardiacly, intramedullarily, intrabone marrow, intradurally, transdermally, intestinally, subcutaneously, sublingually, or topically. The pharmaceutical composition of the present invention can be administered in amounts of 1 to 10,000 mg / kg / day, and can be administered once a day or in several divided doses.

[0058] As used in this invention, the term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, but which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs are articles excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products that have a mild or no direct effect on the human body.

[0059] The quasi-drug composition of the present invention is not particularly limited in terms of its dosage form, and may be a cosmetic composition having a dosage form such as a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a pack, a mask pack, a mask sheet, a gel, or a skin-adhesive type cosmetic, or it may be a transdermal dosage form such as a lotion, ointment, gel, cream, patch, or spray.

[0060] Furthermore, in each formulation, the quasi-drug composition may be arbitrarily selected and blended with other components depending on the formulation of other quasi-drugs or the intended use. The amount of active ingredients mixed can be appropriately determined according to the intended use (suppression or mitigation). For example, it may include common auxiliary agents such as thickeners, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

[0061] The cosmetic composition of the present invention may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, or spray, but is not limited thereto.

[0062] Furthermore, the cosmetic composition of the present invention may further contain fatty substances, organic solvents, solvents, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering agents and chelating agents, and may also contain auxiliary agents commonly used in cosmetics or dermatology, such as preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic surfactants, lipid vesicles, or any other components commonly used in cosmetics. The above components may be introduced in amounts commonly used in dermatology.

[0063] These examples are merely illustrative of the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

[0064] Example 1: Isolation and culture of stem cells In this example, various stem cells were isolated and cultured in order to produce stem cell-derived exosomes.

[0065] Adipose-derived mesenchymal stem cells (AT-MSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), skeletal muscle-derived mesenchymal stem cells (SM-MSCs), cardiomyocyte-derived stem cells (MyoCSCs), peripheral nerve-derived stem cells (PNSCs), synovial membrane-derived mesenchymal stem cells (Syn-MSCs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs) were isolated and cultured using a three-dimensional organ culture method.

[0066] Plasma-derived fibrinogen was dissolved in PBS (phosphate-buffered saline) containing 10 mM CaCl2 at a concentration of 5 mg / mL, and plasma-derived thrombin was dissolved in PBS at a concentration of 1 unit / mL. Adipose tissue (AT), bone marrow (BM), myocardium, skeletal muscle (SM), synovium, peripheral nerve, and umbilical cord (UC) tissues to be impregnated into the hydrogel were cut using a surgical scalpel to a depth of 0.2-2 mm. 3 After cutting the tissue into small sections, hematoma and fibrous tissue were removed from the tissue sections before taking the sections. The tissue sections were washed three times with PBS to remove blood cells from the tissue. After washing, 1 mL of thrombin solution was added per 10 mg of tissue section and placed in an ice bucket for 10 minutes. Then, the same amount of fibrinogen solution was added and mixed uniformly. The mixture was transferred to a culture vessel and left to stand in a 37°C incubator for 1 hour to form a gel. The sections were impregnated with fibrinogen solution to form a fibrin hydrogel with a final wound repair matrix composition of 0.25% fibrinogen and 0.5 unit / mL thrombin.

[0067] The culture medium for culturing organs consisted of 45% (vol / vol) DMEM, 45% Ham's F12, 10% fetal bovine serum (FBS; Invitrogen), 20 ng / mL EGF, 2 ng / mL bFGF, 10 ng / mL IGF, 10 μm / mL gentamicin (gentamicin, Invitrogen), and 200 mg / mL tranexamic acid. Twice the volume of the organ culture medium was added, and after adding the organ culture medium, the culture vessel was placed on an orbital shaker and cultured for 14 days while slowly agitating at a speed of 30 rpm. The organ culture medium was replaced with fresh organ culture medium twice a week.

[0068] After 2 weeks of organ culture, the culture medium was removed, DMEM was added, and the tissue sections were washed three times for 10 minutes each to remove any remaining organ culture medium from the wound repair matrix. After adding fresh organ culture medium without added PAI, the culture vessel was placed on an orbital shaker and stirred at 37°C and 30 rpm for 1 hour to release the stem cells that had migrated and grown in the wound repair matrix into the culture medium. The released stem cells were collected into the culture medium, transferred to a 50 mL centrifuge tube, and centrifuged at 300 × g for 10 minutes. After removing the upper layer, the cells were dispersed in 10 mL of cell culture medium, and the cell count was calculated using a hemocytometer. 2 Stem cells were seeded into culture vessels at a cell density of 3,000 cells per vessel and amplified and cultured in a monolayer culture environment.

[0069] After centrifugation using a ficoll gradient, monocyte cells were separated from the umbilical cord blood. The cells were washed three times by adding PBS and centrifugation. Afterward, the cells were suspended in cell culture medium, seeded, and cultured for two weeks. Colonies formed after culture were isolated after trypsin treatment and amplified using a standard monolayer culture method. All stem cells used for exosome generation were obtained from cells collected after four passages.

[0070] As shown in [Figure 1], cells isolated and cultured from fat, bone marrow, umbilical cord blood, cardiac muscle, skeletal muscle, peripheral nerves, synovial membrane, and umbilical cord all exhibited scaffold-dependent growth by attaching to the attachment vessel, confirming the typical spindle-shaped characteristics of stromal cells.

[0071] Example 2: Analysis of the immune expression characteristics of stem cells The immunoexpression characteristics of stem cells amplified and cultured using a standard monolayer culture method after organ culture were analyzed. 100,000 cells were reacted with fluorescently labeled anti-human antibodies against CD31, CD34, CD44, CD45, CD73, and CD105. The positive rate for each antibody was analyzed using a FACSCalibur (Becton Dickinson, USA) flow cytometer (FCM), and expression rates were analyzed for more than 100,000 cells.

[0072] [Table 1]

[0073] As shown in [Figure 2] and [Table 1], more than 90% of the scaffold-dependent cells isolated and cultured from adult tissue positively expressed the stromal cell labels CD44, CD73, and CD105. However, less than 2% expressed vascular endothelial cells, hematopoietic cells, and the immunocytoplasmic cell labels CD31, CD34, and CD45, indicating that the cells used for exosome generation had a purity of more than 90% stromal cells.

[0074] Example 3: Confirmation of differentiation characteristics of stem cells into adipocytes and osteoblasts To evaluate the differentiation ability of stem cells into adipocytes, 200,000 cells were seeded into 24-multiwell tissue culture plates and cultured for 14 days in a medium consisting of 90% DMEM supplemented with 10% CS, 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma), 80 μM indomethacin (Sigma), 1 μM dexamethasone (Sigma), and 5 μm / mL insulin (Sigma). Differentiation of cells into adipocytes was determined by staining with 0.5% Sudan Black (Sigma) solution, an indicator of intracellular lipid accumulation, at room temperature for 1 hour, and observing the presence or absence of lipid accumulation in the cytoplasm.

[0075] To evaluate the differentiation ability of stem cells into osteoblasts, 200,000 cells were seeded into 24-multiwell tissue culture plates and cultured for 14 days in a medium consisting of 90% α-MEM supplemented with 10% CS, 50 μm / mL ascorbic acid, 0.1 μM dexamethasone (Sigma), and 1 mM calcium glycerophosphate (Sigma). Differentiation of stem cells into osteoblasts was assessed by staining mineral accumulation with 1 μM calcein and then examining the cells under a microscope.

[0076] As shown in [Figure 3], differentiation from anchorage-dependent stem cells into adipocytes and osteoblasts was observed. Differentiation into adipocytes was confirmed by the accumulation of multiple vesicles in the cytoplasm, and the accumulation of lipid vesicles in the cytoplasm, which was positive for Sudan Black, confirmed the differentiation into adipocytes.

[0077] During differentiation into osteoblasts, mineral crystals were observed in the extracellular matrix using a phase-contrast microscope, and these minerals were confirmed to be calcium phosphate using a calcein fluorescence microscope.

[0078] Although there were differences in the degree of differentiation into adipocytes or osteoblasts depending on the tissue of origin, differentiation into adipocytes and osteoblasts was confirmed in all stem cells, confirming that they are stem cells with multipotency.

[0079] Example 4: Structural adjustment of hydrogels according to polymer concentration After dissolving fibrinogen (Sigma) in DMEM, fibrinogen solutions of four different concentrations—2.5, 5.0, 10.0, and 20.0 mg / mL—were prepared. 100 μm / mL of aminomethylbenzoic acid (Sigma) was added to the fibrinogen solutions. After dissolving thrombin (Sigma) in DMEM, a 1 unit / mL thrombin solution was prepared. To confirm the size and connectivity of fibrin pores, a fibrinogen solution was prepared by mixing Alexa Fluor 546-conugated fibrinogen (Molecular Probes) in a 1:10 ratio. This solution was then mixed with the 1 unit / mL thrombin solution in a 1:1 ratio, and a fibrin hydrogel was prepared by polymerization and crosslinking reactions at 37°C for 1 hour. The structural properties of fibrin hydrogels generated at four different fibrinogen concentrations (1.25, 2.5, 5.0, and 10.0 mg / mL) were evaluated using a confocal microscope (Zeiss LSM 900).

[0080] As shown in [Figure 4], the size of the pores in the manufactured fibrin hydrogel decreased as the concentration of the constituent polymer, fibrinogen, increased, forming a dense, porous hydrogel structure.

[0081] On the other hand, as the fibrinogen concentration decreased, the pore size decreased, and a hydrogel with a coarser structure was formed. As shown in [Figure 4B], the pore sizes of fibrin hydrogels prepared with four concentrations of fibrinogen solution (1.25, 2.5, 5.0, and 10.0 mg / mL) were 265.7, 165.2, 76.8, and 38.5 nm, respectively, and the pore diameter decreased significantly as the fibrinogen concentration increased, exhibiting faithful structural characteristics.

[0082] Example 5: Cell elongation using hydrogel nanostructures The interaction between hydrogels and stem cells based on their physical and structural properties was evaluated by 3D culture of cardiomyocyte-derived stem cells (MyoCSCs).

[0083] 1 mL of fibrinogen solution at four concentrations (1.25, 2.5, 5.0, and 10.0 mg / mL) was mixed with 1 million MyoCSCs, and then each was mixed with the same volume of thrombin solution. Polymerization and cross-linking reactions were carried out at 37°C for 1 hour to prepare fibrin hydrogels impregnated with MyoCSCs. A serum-free medium containing 50 μm / mL ascorbic acid and 100 μm / mL N-acetyl cysteine ​​was prepared as the cell culture medium, and the prepared cell culture medium was added to the fibrin impregnated with MyoCSCs and cultured for 12 hours.

[0084] After culturing, the hydrogel impregnated with stem cells was fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, it was reacted with Alexa Fluor 488-conugated phalloidin (Invitrogen) for 60 minutes, and the degree of cell elongation was evaluated by measuring the length of the microfibers formed in the cytoplasm using a confocal microscope.

[0085] As shown in [Figure 5], the degree of cell elongation varied depending on the type of fibrin hydrogel due to the microfibers within the cytoplasm. Stem cells in the hydrogel exhibited different shapes from those in the 2D culture environment; cell elongation could be observed in the 3D axial plane, and the cytoplasm was formed in a long and narrow manner.

[0086] The degree of cell elongation of stem cells in coarse fibrin hydrogels formed with low concentrations of fibrinogen was high, while cell elongation was significantly reduced in hydrogels formed with high concentrations of fibrinogen. As shown in [Figure 5B], the cell elongation length was highest in fibrin hydrogels with a coarse structure. The cell elongation lengths of stem cells in fibrin hydrogels prepared with four concentrations of fibrinogen (1.25, 2.5, 5.0, and 10.0 mg / mL) were 112.5, 75.0, 25.2, and 7.4 μm, respectively. The significantly higher microfiber lengths observed in hydrogels with a coarse structure confirmed that physical cell elongation can be regulated by the structural and physical properties of the hydrogel.

[0087] Example 6: Activation of stem cell signaling by hydrogel nanostructures The interaction between hydrogels and stem cells based on their physical and structural properties was evaluated by 3D culture of cardiomyocyte-derived stem cells (MyoCSCs).

[0088] After preparing a hydrogel impregnated with stem cells using the same method as in Example 5 above, 4% formaldehyde was added and fixed for 2 hours. After washing three times with PBS, the hydrogel was reacted with β-integrin (Cell Siganling Technology) and p-FAK (cell Signaling Technology) primary antibody at 37°C for 1 hour, and then reacted with Alexa Fluor 488-conugated anti-mouse IgG (Invitrogen) for 30 minutes.

[0089] Nuclei were stained with DAPI (diamidino-2-phenylindole, Molecular Probes), and the expression levels of β-integrin and p-FAK were evaluated by confocal microscopy. Western blotting was performed to evaluate integrin-mediated cell signaling pathways. Intracellular proteins were extracted in a 3D hydrogel or 2D culture environment, followed by PAGE electrophoresis. After blotting the PAGE gel onto an Immobilon (Sigma) membrane, it was reacted with primary antibodies for p-FAK (Cell Signaling Technology), p-JNK (Cell Signaling Technology), and p-β-catenin (Cell Signaling Technology) for 1 hour, followed by reaction with an AP-conjugated secondary antibody for 30 minutes. Subsequently, it was reacted with an ECL (ThermoFisher Scientific) substrate, and band intensity was measured. The results were compared after correcting for β-actin expression intensity.

[0090] As shown in [Figure 6], we were able to confirm differences in the expression of β-intecrin, p-FAK, and β-catenin in stem cells impregnated in a 3D environment based on the hydrogel structure characteristics. Compared to a 2D culture environment, the expression of p-FAK, p-JNK, and β-catenin was significantly increased in the 3D culture environment. In particular, there was an inverse relationship with the concentration of the hydrogel constituent polymer, and the expression rates of p-FAK, p-JNK, and β-catenin increased with a coarser hydrogel structure. In the case of P-FAK, the expression rate increased 85.4, 67.8, 34.2, 34.2, and 0.7 times in hydrogel-infused stem cells with fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg compared to the 2D culture environment. In the case of P-JNK, the expression rate increased 35.4, 25.4, 12.6, and 0.8 times in hydrogel-infused stem cells with fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg compared to the 2D culture environment, while the expression rate of p-β-catenin increased 12.5, 9.4, 3.5, and 0.9 times.

[0091] These results confirm that microfibrillation increases with the degree of cell elongation, and that p-FAK, p-JNK, and β-catenin expression also increases, thus confirming that cell elongation can activate cellular signaling pathways.

[0092] Example 7: Characteristics of mRNA expression in the Wnt signaling pathway of stem cells in 2D and 3D culture environments In this example, the Wnt signaling pathway-related mRNA expression rate of stem cells in a 2D culture or 3D hydrogel culture environment was measured using a PCR microarray kit (RT 2 Evaluation was performed using a Profiler PCR Array (CureZen).

[0093] Total RNA was extracted from MyoCSCs cultured in a 2D environment using TRIzol (Invitrogen), and then cDNA was synthesized using the reverse transcriptase provided in the kit.

[0094] The 3D hydrogel was prepared using 1.25 mg / mL fibrinogen and 0.5 unit / mL thrombin solution, and 1.0 × 10⁻¹⁶ units were added during hydrogel preparation. 6 MyoCSCs were impregnated and cultured. After 3 days of culture, total RNA and cDNA were synthesized using the same method as in the 2D culture environment.

[0095] The expression rates of p-FAK, p-JNK, and p-β-catenin were confirmed. RT 2 The expression of 82 genes, including Wnt ligands, receptors, and subtarget genes, filed using a Profiler PCR Array (CureZen), was evaluated. Expression rates were corrected for housekeeping genes and expressed as percentages compared to MyoCSCs in a 2D culture environment using the ΔΔCT method.

[0096] As shown in [Figure 8], the expression of Wnt ligands, receptors, and Wnt target submRNAs was significantly increased in the 3D hydrogel environment. The Wnt signaling pathway was activated by extracellular physical stimuli, and in particular, the degree of microfibrillation of actin filaments in the cytoplasm increased the Wnt signaling pathway.

[0097] As demonstrated in this embodiment, we were able to induce physical stimulation of stem cells in a 3D hydrogel culture environment, resulting in the promotion of actin filament tissue, a significant increase in microfiber formation as a result of cell elongation, and ultimately, activation of the Wnt signaling pathway, which we confirmed at the mRNA level.

[0098] Compared to the 2D culture environment, the expression rate of Wnt ligand mRNA increased 56.6 times for WNT1, 32.3 times for WNT10A, 15.4 times for WNT10B, 17.0 times for WNT11, 77.6 times for WNT2B, 27.5 times for WNT3A, and 47.0 times for WNT6 (p<0.05). Wnt receptor mRNA was also increased and expressed in stem cells in the 3D culture environment.

[0099] Stem cell mRNA expression in the 3D environment increased significantly compared to the 2D culture environment: FZD1 increased 16.4 times, FZD10 57.9 times, FZD3 11.0 times, FZD5 7.3 times, FZD9 14.0 times, and SFRP2 71.7 times. As a result, significant changes were also observed in the expression of target genes that are subordinately affected by the Wnt signaling pathway.

[0100] Compared to stem cells cultured in a 2D environment, CCND1 increased 12.2 times, CCND2 7.5 times, CD44 3.0 times, CDX1 90.2 times, CDX2 63.1 times, FGF4 47.7 times, FGF9 62.5 times, FN1 6.7 times, MYC 29.8 times, MYCN 45.0 times, NANOG 49.3 times, SOX2 84.1 times, and VEGFA 87.9 times. These results suggest that 3D hydrogel structure modification can activate the Wnt signaling pathway in stem cells, thereby increasing functional gene expression.

[0101] Example 8: 3D hydrogel nanostructure and the generation of stem cell exosomes associated with cell elongation We evaluated the relationship between the structural properties of hydrogels and stem cell elongation, p-FAK, and exosome generation.

[0102] After preparing a hydrogel impregnated with stem cells using the same method as in Example 5 above, 4% formaldehyde was added and fixed for 2 hours. After washing three times with PBS, the hydrogel was reacted with p-FAK (cell Signaling Technology) primary antibody at 37°C for 1 hour, and then reacted with Alexa Fluor 488-conugated anti-mouse IgG (Invitrogen) for 30 minutes. Exosome labels anti-CD9, CD63, and CD81 were reacted with the label (Thermo Fisher Scientific) for 1 hour, and then reacted with Alexa Fluor 594 (Molecular Probes). The nuclei were stained with DAPI (diamidino-2-phenylindole, Molecular Probes). p-FAK expression and cytoplasmic exosome production rate were evaluated by confocal microscopy.

[0103] [Table 2]

[0104] As shown in [Figure 9], the level of p-FAK expression in stem cells within the 3D hydrogel increased proportionally to exosome generation. p-FAK expression increased in proportion to cell elongation, and the levels of p-FAK expression significantly increased the levels of CD9, CD63, and CD81-positive factors, which are exosome labels in the cytoplasm. Similar to previous results, the intensity of exosome-positive factors significantly increased in cells within a coarse hydrogel structure produced with low fibrinogen concentration. These results confirm that p-FAK expression can be regulated by the hydrogel structure, and consequently, stem cell exosome generation can be regulated.

[0105] Example 9: Exosome secretion into cell culture medium using 3D hydrogel nanostructures We evaluated the relationship between hydrogel structural properties, cell elongation of cardiomyocyte-derived stem cells, and the rate of exosome secretion into the cell culture medium.

[0106] 1 mL of fibrinogen solution at four concentrations (1.25, 2.5, 5.0, and 10.0 mg / mL) was mixed with 1 million MyoCSCs, and then each was mixed with the same volume of thrombin solution. Polymerization and cross-linking reactions were carried out at 37°C for 1 hour to prepare fibrin hydrogels impregnated with MyoCSCs. A serum-free medium containing 50 μm / mL ascorbic acid and 100 μm / mL N-acetylcysteine ​​in DMEM / F12 was prepared as the cell culture medium. The prepared cell culture medium was added to the fibrin impregnated with MyoCSCs and cultured for 24 hours.

[0107] Exosomes were isolated from the cell culture medium collected after 24 hours using the Tangential flow filtration (TFF) method, utilizing a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The number of nanoparticles in the culture medium of the concentrated and purified exosomes was evaluated using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical). Protein content within the exosomes was measured using a microBCA (Fisher Thermo Scientific). After culturing the same MyoCSCs in a 2D culture environment for 24 hours, exosomes were isolated from the resulting cell culture medium.

[0108] [Table 3]

[0109] As shown in [Figure 10], significant differences in the number of exosomes generated and secreted from stem cells were observed depending on the structural properties of the hydrogel. The number of exosomes secreted into the cell culture medium from stem cells cultured in a coarse hydrogel manufactured with a low high molecular weight was the highest. The number of exosomes in fibrin hydrogels manufactured according to four concentrations of fibrinogen (1.25, 2.5, 5.0, and 10.0 mg / mL) was 25.4 × 10⁶. 9 / mL, 16.1 × 10 9 / mL, 5.7 × 10 9 / mL, 1.1 × 10 9 At a concentration of fibrinogen at low levels / mL, the number of exosomes was significantly higher in coarse-structured fibrin hydrogels.

[0110] Similar to the number of exosomes, the protein content was significantly higher in stem cells secreted in hydrogels with a coarser structure. The protein content in fibrin hydrogels prepared at fibrinogen concentrations of 1.25, 2.5, 5.0, 5.0, and 10.0 mg / mL was 318.5, 213.1, 68.4, and 18.5 μm / mL, respectively. The protein content generated and isolated from stem cells as a cell culture medium was significantly higher in fibrin hydrogels with a coarser structure. It was confirmed that stem cells cultured in fibrin hydrogels (excluding the 10.0 mg / mL concentration) supported significantly higher exosome secretion compared to stem cells cultured in a 2D culture environment.

[0111] Example 10: Confirmation of exosome size generated in 2D culture and 3D hydrogel culture environments In this example, we tested the structural differences of exosomes produced under different culture environments.

[0112] Cardiac muscle-derived stem cells were impregnated into a 3D fibrin hydrogel and cultured according to the method described in <Example 9> above, and exosomes were isolated. The size of nanoparticles in the culture medium of concentrated and purified exosomes was measured using a nanoparticle tracking analyzer (NanoSight Pro, Malvern Panalytical). After culturing the same MyoCSCs in a 2D culture environment for 24 hours, exosomes were separated from the resulting cell culture medium, and the size of the nanoparticles was measured using the same method.

[0113] As shown in [Figure 11], no difference in exosome particle size was observed depending on the culture environment. The average diameters of exosomes produced in gels with fibrinogen concentrations of 1.25, 2.5, 5.0, 5.0, and 10.0 mg / mL were 118, 116, 117, and 121 nm, respectively, and no difference was observed compared to the average diameter of exosomes produced in a 2D environment (121 nm). There was also no difference in the Mode value, which represents the size of the most frequently occurring nanoparticles, between the 2D and 3D culture environments, and no difference was observed due to the concentration of the hydrogel constituent polymer.

[0114] Example 11: Exosome immunoexpression characteristics of exosomes generated and secreted from stem cells under 2D culture and 3D hydrogel culture conditions The immunoexpression characteristics of exosomes generated and isolated from cardiomyocyte-derived stem cells in 2D and 3D culture environments were analyzed using multiplex bead kits (MACSPlex Exosome Kit, Mitenyl Biotec). Cardiac muscle-derived stem cells were impregnated into a 3D fibrin hydrogel and cultured according to the method described in <Example 9> above, and exosomes were isolated.

[0115] Exosomes were centrifuged at 2,500 × g for 15 minutes, and the supernatant was collected and analyzed. 30 μL of exosomes were taken and mixed with MACSPlex buffer in a 1:1 ratio. 8 μL of MACSPlex exosome capture beads were added to each well of a MACSPlex 96-well plate, followed by 60 μL of exosomes. After reacting at room temperature for 30 minutes, the immunoexpression characteristics of the exosomes were analyzed using flow cytometry, and the degree of expression was determined by the expression intensity (median APC-A).

[0116] [Table 4] TIFF2026512224000006.tif135170

[0117] As shown in [Table 4] and [Figure 12], there was no difference in the immunoexpression characteristics of exosomes generated by different culture environments. Exosomes generated in both 2D and 3D culture environments showed high expression levels of the exosome labels CD9, CD63, and CD81, but no difference was observed depending on the culture environment. Similarly, no difference was observed in the expression of the cardiomyocyte-derived stem cell labels CD105, CD44, and CD29 between the 2D and 3D culture environments, and all exosomes showed high expression levels.

[0118] Example 12: Exosome generation rate according to stem cell type in 2D and 3D culture environments In this example, the yield of exosomes produced by changing the culture environment according to the type of stem cell was analyzed. Stem cells derived from fat, bone marrow, umbilical cord blood, synovial membrane, umbilical cord, adult stem cells derived from peripheral nerves, and myocardial cells were cultured in 2D and 3D environments.

[0119] Each of the stem cells described above was impregnated into a 3D fibrin hydrogel and cultured according to the method shown in <Example 9> above, after which exosomes were isolated. The exosome production rate of the concentrated and purified exosomes was measured using a nanoparticle tracking analyzer (NanoSight Pro, Malvern Panalytical).

[0120] As shown in [Figure 13], a significant difference in exosome production was observed depending on the culture environment. Compared to the 2D culture environment, the number of exosomes produced in the 3D culture environment increased significantly by 1.7 to 2.9 times. This significant increase was confirmed in all mesenchymal stem cells derived from cardiomyocytes (MyoCSCs), peripheral neural stem cells (PNSCs), umbilical cord blood (UC-MSCs), and bone marrow (BM-MSCs) compared to the 2D culture environment.

[0121] Example 13: Exosome generation rate based on the number of cultured cells in a 3D fibrin hydrogel culture environment In this example, the exosome production yield was analyzed according to the culture density of stem cells.

[0122] 2, 10, 20, 200, 1,000, 2,000, 2,000 × 10 per 1 mL of thrombin solution 4 After mixing with the cells, each was combined with a 2.5 mg / mL fibrinogen solution, and then impregnated into a 3D fibrin hydrogel according to the method described in <Example 9> above. The cells were cultured, and exosomes were isolated.

[0123] The total number of exosomes in the concentrated and purified exosomes was measured using a nanoparticle tracking analyzer (NanoSight Pro, Malvern Panalytical).

[0124] As shown in [Figure 14], the exosome generation rate increased in proportion to the number of cells impregnated into the hydrogel. When 10,000, 50,000, 100,000, 1,000,000, 5,000,000, and 10,000,000 cells were impregnated per mL of hydrogel and cultured, the exosome generation rates were 2.6, 4.3, 8.5, 25.4, 32.9, and 12.5 × 10⁶. 9 A number of exosomes were generated.

[0125] In the case of fibrin hydrogel, up to 5 million cells can be cultured per mL, and in this case, 3.3 × 10⁶ cells per mL of cell culture medium. 10 Exosomes were successfully generated. However, when the cell count exceeded 10 million cells per mL, a phenomenon of hydrogel contraction occurred due to the cells. As a result, the hydrogel fell out of the culture vessel, compressing the cell-mediated hydrogel and reducing the exosome generation rate.

[0126] In a 2D culture environment, cm 2 The maximum number of cells that can be cultured per unit area varies depending on the type of stem cell, but it is possible to culture 10,000 to 50,000 cells. However, in a 3D culture environment, cm 2It was confirmed that this high-density culture method allows for culturing cell counts from 10,000 to 5 million cells per unit area, enabling high-density culture, and that the number of exosomes that can be produced significantly increases with increasing cell counts per unit area.

[0127] Example 14: Exosome generation rate by 3D hydrogel polymer components In this example, the correlation between the generation of stem cell microfibers and the exosome generation rate within the hydrogel, depending on the type of hydrogel constituent polymer, was investigated. Hydrogels containing fibrin, collagen, gelatin, hyaluronic acid, and chondroitin were applied as 3D cell culture substrates, and the exosome generation rate was investigated.

[0128] 2 × 10 per 1 mL of crosslinking agent solution 6 After suspending with cells and mixing in 1:1 ratios with 2.5 mg / mL fibrinogen, 0.2% collagen, 0.4% gelatin, 0.2% hyaluronic acid, and 0.2% chondroitin solution, polymerization and cross-linking reactions are carried out at 37°C for 1 hour, resulting in 1 × 10¹⁶ hydrogels per mL. 6 We manufactured fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels, each impregnated with cardiomyocyte-derived stem cells.

[0129] A serum-free medium containing 50 μg / mL ascorbic acid and 100 μg / mL N-acetylcysteine ​​was prepared as the cell culture medium. The prepared cell culture medium was added to fibrin impregnated with MyoCSCs and cultured for 24 hours.

[0130] Exosomes were separated from the cell culture medium collected after 24 hours using the Tangential flow filtration (TFF) method, utilizing a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The total number of exosomes in the concentrated and purified exosomes was measured using a nanoparticle tracking analyzer (NanoSight Pro, Malvern Panalytical).

[0131] As shown in [Figure 15], the lengths of microfibrils of cardiomyocyte-derived stem cells impregnated in fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels were 112.5, 78.5, 63.4, 58.9, and 61.8 μm, with fibrin hydrogel showing the highest length. The length of microfibrils of cardiomyocyte-derived stem cells in the hydrogel showed a proportional relationship with the exosome generation rate.

[0132] Exosome generation from cardiomyocyte-derived stem cells within fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels was 25.4, 12.7, 8.5, 6.4, and 7.2 × 10⁻¹⁴. 9 In this study, the exosome generation rate of cardiomyocyte-derived stem cells within fibrin and collagen hydrogel was significantly higher.

[0133] The results above confirm that the cell elongation of stem cells impregnated in hydrogel is altered by the hydrogel's polymer components, and that the length of the microfibers, which reflect cell elongation, is proportionally related to the exosome production rate.

[0134] Example 15: Exosome generation rate in a 3D synthetic polymer and copolymer hydrogel culture environment In this example, a 3D culture environment was constructed using a hydrogel of synthetic polymer components and a hydrogel copolymerized with synthetic polymer and fibrin. Cardiac muscle-derived stem cells were then cultured, and the exosome generation rate was analyzed depending on the type of hydrogel.

[0135] 2 × 10⁶ per 1 mL of thrombin or crosslinking agent solution 6 After mixing with cells and then mixing in a 1:1 ratio with a 2.5 mg / mL fibrinogen solution or a 5% PGA, PEG, PLGA, PLLA polymer solution, polymerization and crosslinking reactions are carried out at 37°C for 1 hour, resulting in 1 × 10⁶ per mL of hydrogel. 6 We manufactured fibrin and PGA, PEG, PLGA, and PLLA hydrogels impregnated with cardiomyocyte-derived stem cells.

[0136] To enhance the cytocompatibility of synthetic polymer hydrogels, hydrogels copolymerized with fibrin were fabricated and used as 3D substrates. 2 × 10⁶ units were used to impregnate the fibrin-synthetic polymer copolymerized hydrogel with cardiomyocyte-derived stem cells. 6 Cardiac stem cells are mixed with a crosslinking agent and a solution containing thrombin, then mixed in 1:1 ratios with 5% PGA, PEG, PEG, PLGA, and PLLA polymer solutions containing 2.5 mg / mL fibrinogen solution, followed by polymerization and crosslinking reactions at 37°C for 1 hour, resulting in 1 × 10⁶ cells per mL. 6 Cardiac-derived stem cells were impregnated into a hydrogel copolymerized with fibrin, PGA, PEG, PLGA, and PLLA.

[0137] A serum-free medium containing 50 μg / mL ascorbic acid and 100 μg / mL N-acetylcysteine ​​was prepared as the cell culture medium. The prepared cell culture medium was added to fibrin impregnated with MyoCSCs and cultured for 24 hours.

[0138] Exosomes were separated from the cell culture medium collected after 24 hours using the Tangential flow filtration (TFF) method, utilizing a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The total number of exosomes in the concentrated and purified exosomes was measured using a nanoparticle tracking analyzer (NanoSight Pro, Malvern Panalytical).

[0139] As shown in [Figure 16], the degree of microfibril formation of cardiomyocyte-derived stem cells in synthetic polymer hydrogels was at 1 / 10th the level compared to fibrin hydrogels, and low microfibril formation was confirmed with lengths of 13.5, 12.7, 14.6, and 13.7 μm for cardiomyocyte-derived stem cell microfibrils in hydrogels containing PGA, PEG, PLGA, and PLLA components.

[0140] However, in the case of hydrogels mixed with synthetic polymers and fibrin hydrogels, the lengths of myocardial-derived stem cell microfibrils were 42.5, 58.9, 85.4, and 65.8 μm. Cell compatibility improved through fibrin mixing, and the degree of microfibril formation, which indicates cell elongation, significantly increased with the increase in ligands that bind to the hydrogel substrate and cells.

[0141] The rate of exosome generation from cardiomyocyte-derived stem cells in hydrogels containing synthetic polymers, or a mixture of synthetic polymers and fibrin, was significantly improved in the hydrogel containing a mixture of synthetic polymers and fibrin. For the synthetic polymers PGA, PEG, PLGA, and PLLA, the number of exosomes generated per mL was 2.5, 1.7, 1.8, and 2.8 × 10⁶, respectively. 9 It showed a low generation rate.

[0142] In the case of synthetic polymer hydrogels mixed with fibrin, the exosome generation rate increased, and when PGA, PEG, PLGA, and PLLA polymer hydrogels mixed with fibrin were cultured as 3D substrates, the exosome generation rates were 18.5, 19.7, 16.7, 16.7, and 12.8 × 10⁻⁶.9 The result showed a significant increase compared to the synthetic polymer hydrogel before mixing.

[0143] The results above confirm that cellular microfiber formation is related to ligands that can bind to cells within the hydrogel, that the degree of microfiber formation is closely related to the exosome generation rate from stem cells, and that in the case of synthetic polymer hydrogels, cell compatibility decreases, resulting in a decrease in microfiber formation and exosome generation rate.

[0144] Example 16: miRNA expression in stem cell-derived exosomes generated in 2D and 3D hydrogel culture environments In this example, to analyze the characteristics of exosomes generated by the culture environment, the expression level of miRNA, one of the main mediators of exosome action, was measured.

[0145] Cardiac-derived stem cells were cultured in a 3D fibrin hydrogel according to the method described in <Example 9> above, and then exosomes were isolated. The exosomes were centrifuged at 2,500 × g for 15 minutes, and miRNA was isolated from the upper layer using a miRNA isolation kit (miRNA isolation kit, mirvana miRNA isolation kit, Invitrogen). A miRNA library was prepared from the miRNA extracted from the exosomes by reverse transcriptase reaction (Taqman MicroRNA Reserve Transcription Kit, Invitrogen). Quantitative PCR was performed using a miRNA-specific target probe (Affymatrix) according to the Taqman method (TaqMan MicroRNA Assay Kit).

[0146] As shown in [Figure 16], we confirmed that exosomes produced in the 3D hydrogel culture environment of the present invention showed a significant increase in the content of miRNAs that act on angiogenesis, tissue regeneration, and inflammation regulation, namely let-7b-3b, miR126-5p, miR145-5p, miR146a-5p, and miR185-5p, compared to exosomes obtained in a 2D culture environment. In other words, we confirmed that not only can exosome production be increased through the 3D hydrogel culture of the present invention, but the composition of active ingredients within the exosomes can also be increased, thereby enhancing the efficacy of angiogenesis, tissue regeneration, and inflammation regulation.

[0147] Example 17: Wound repair, angiogenesis, and anti-inflammatory effects of stem cell-derived exosomes generated in 2D and 3D hydrogel culture environments In this example, to analyze the differences in the effects of stem cell-derived exosomes generated by different culture environments, we tested the effects on the growth of fibroblasts involved in wound repair, the growth of vascular endothelial cells involved in angiogenesis and vascular elongation, and the isolation of inflammatory cytokines secreted from inflammatory cells.

[0148] Mesenchymal stem cells derived from adipose tissue, bone marrow, and muscle, stem cells derived from peripheral nerves, and stem cells derived from cardiomyocytes were each impregnated into 3D fibrin hydrogel and cultured according to the method described in <Example 9> above, and then exosomes were isolated.

[0149] The effects of exosomes were evaluated on a cell basis using fibroblasts (3T3-L1), human umbilical cord vein endothelial cells (HUVECs), and RAW264.7 inflammatory cells. To assess the effect of exosomes on inducing cell growth, DFs and HUVECs cells were seeded at 5,000 cells per well in a 96-well plate, and 1.0E+08 exosomes were added to each well. The cells were then cultured for 24 hours. After culturing, the cells were lysed in 0.1% SDS / TE solution, and the growth rate compared to cells without exosome administration was analyzed as a ratio (fold) using the PicoGreen dsDNA quantification kit.

[0150] Vascular tube growth was evaluated by first producing microspheres composed of 300 HUVEC cells, then impregnating and culturing these microspheres in a collagen gel, and measuring the increased length of vascular tubes with and without exosome addition.

[0151] 100,000 RAW264.7 inflammatory cells were seeded in a 24-well plate and then sensitized with 10 μg LPS (lipopolysaccharide, Sigma). Exosomes were added during LPS sensitization, and the concentrations of TNF-α and IL-β secreted from LPS-sensitized inflammatory cells were analyzed using an ELISA kit (R&D Systems).

[0152] As a result of treating 3T3-L1 cells with the exosomes generated above, it was confirmed that 3T3-L1 cell growth was significantly increased by stem cell-derived exosomes produced in the 3D hydrogel culture environment of the present invention compared to the 2D culture environment, as shown in [Figure 18]. When stem cell exosomes derived from fat, bone marrow, cardiac muscle, peripheral nerve, and muscle cells generated in the 3D culture environment were treated, cell growth increased by 28%, 31%, 119%, 28%, and 36% compared to exosomes generated in the 2D culture environment, confirming a high wound repair effect.

[0153] When HUVEC cells were treated with exosomes generated in a 3D culture environment, the results showed a significantly higher induction of HUVEC cell growth, as shown in [Figure 19]. When adipose, bone marrow, cardiac muscle, peripheral nerve, and muscle-derived stem cell exosomes generated in a 3D culture environment were treated, they induced HUVEC growth by 10%, 18%, 56%, 50%, and 38% compared to exosomes generated in a 2D culture environment.

[0154] Figure 20 shows the growth rate of vascular tubules formed in HUVECs treated with exosomes generated in a 3D culture environment. The lengths of HUVEC vascular tubules from adipose, bone marrow, cardiac muscle, peripheral nerve, and muscle-derived stem cell exosomes generated in a 3D culture environment were 3.1, 2.8, 4.5, 3.5, and 4.9 mm, respectively. This was significantly higher than the lengths of exosomes generated in a 2D culture environment (2.7, 2.3, 3.8, 2.8, and 3.5 mm), demonstrating a significantly higher ability to induce vascular tubule growth in exosomes generated in a 3D culture environment. The results showing that stem cell exosomes generated in a 3D culture environment promote the growth of vascular endothelial cells and vascular tubules suggest a high angiogenic effect.

[0155] As shown in Figures 21 and 22, after treating RAW264.7 cells with exosomes generated in a three-dimensional culture environment, the secretion of inflammatory cytokines TNF-α and IL-β, which are secreted after sensitization with LPS, decreased compared to exosomes generated in a two-dimensional culture environment.

[0156] A significant decrease in TNF-α secretion was observed with exosomes generated in a 3D culture environment. When bone marrow, adipose tissue, cardiac muscle, peripheral nerve, and muscle-derived stem cell exosomes were treated, the TNF-α levels were 302, 364, 213, 306, and 254 pg / mL, respectively, which were lower than those observed when exosomes generated in a 2D culture environment were treated.

[0157] When RAW264.7 cells were treated with exosomes generated in a 3D culture environment, the IL-1β secretion inhibitory ability was also higher. Treatment with bone marrow, adipose tissue, cardiac muscle, peripheral nerve, and muscle-derived stem cell exosomes resulted in secretion levels of 330, 365, 245, 245, 305, and 287 pg / mL in cells treated with exosomes generated in a 2D culture environment. In contrast, the levels were 405, 421, 356, 364, and 358 pg / mL in cells treated with stem cell exosomes generated in a 2D culture environment.

[0158] The wound repair, angiogenesis, and anti-inflammatory effects of the cells described above were significantly higher in stem cell exosomes in a 3D culture environment, confirming that the culture environment can enhance the effects of exosomes.

[0159] We have confirmed that exosomes produced by the method of the present invention increase miRNA components that can regulate angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced by the method of the present invention can be usefully utilized as compositions for angiogenesis, tissue regeneration, or anti-inflammatory purposes.

Claims

1. (a) A step of mixing a hydrogel in a sol state, which contains a polymer chain having ligands that can bind to cellular integrins and capable of sol-gel phase transition, with stem cells; and, (b) The step of inducing a phase transition of the hydrogel to a gel state to produce a 3D hydrogel in which stem cells are uniformly collected in three dimensions, and culturing the stem cells; A method for increasing stem cell-derived exosome production by transmitting physical stimuli through cell stretching.

2. The method according to claim 1, characterized in that, without the use of physical devices, the ligand of the hydrogel and the cell directly bind to each other, causing the cell to stretch.

3. The method according to claim 1, characterized in that the hydrogel having a ligand capable of binding to the integrin is a hydrogel obtained by copolymerizing a natural polymer substance selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin, or a synthetic polymer substance selected from the group consisting of the natural polymer substance and PEG (Polyethylene Glycol), PLA (Polylactic Acid), PLGA (Polylactic Glycolic Acid), and PGA (Polyglycolic Acid).

4. The method according to claim 1, characterized in that step (b) is a crosslinking agent added to the mixture of step (a) to cause a phase transition of the hydrogel to a gel state.

5. The method according to claim 1, characterized in that, in order to adjust the physical strength of the 3D hydrogel in step (b), one or more polymeric substances selected from the group consisting of PEG, PLGA, PGA, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid are further mixed into step (a).

6. The method according to claim 1, characterized in that, in order to suppress cellular degradation of the 3D hydrogel in step (b) and enhance its stability, one or more MMP inhibitors selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat, which can inhibit matrix metalloproteinases (MMPs) selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9, are further mixed in step (a).

7. The method according to claim 1, characterized in that, in order to suppress cellular degradation of the 3D hydrogel in step (b) and enhance its stability, one or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid are further mixed in step (a).

8. The method according to claim 1, characterized in that the 3D hydrogel of step (b) has a porous structure of 50 nm to 300 nm size and has structural properties that provide cell-cell junctions and promote cell stretching.

9. The method according to claim 1, characterized in that the stem cells cultured in step (b) have a morphology that extends along a three-dimensional axial plane, and the cells can extend to a length of 15 μm to 400 μm.

10. 1.0 × 10 per mL of hydrogel 4 ~5.0 x 10 6 The method according to claim 1, characterized in that stem cells can be impregnated at high density and cultured.

11. The method according to claim 1, characterized in that, in step (b) above, the stem cells cultured through the modification of the hydrogel physicochemical properties and structural characteristics have the integrin-FAK-JNK-β-catenin signaling pathway activated.

12. The method according to claim 1, characterized in that, in step (b) above, the stem cells cultured through the modification of hydrogel properties and structural characteristics have an activated Wnt signaling pathway and increased Wnt signaling subtarget gene expression.

13. The method according to claim 1, characterized in that the stem cell-derived exosomes produced by the above method have an increased composition of one or more angiogenesis-regulating miRNAs selected from the group consisting of let-7b-3b and miR126.

14. The method according to claim 1, characterized in that the stem cell-derived exosomes produced by the above method have an increased composition of one or more inflammation-modulating miRNAs selected from the group consisting of miR145-5p, miR146a-5p, and miR185-5p.

15. The method according to claim 1, wherein the stem cells may be adult stem cells or mesenchymal stem cells, and are preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, cardiomyocyte-derived stem cells, muscle-derived stem cells, peripheral nerve-derived stem cells, synovial membrane-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

16. A composition for angiogenesis, tissue regeneration, or anti-inflammatory purposes, comprising stem cell-derived exosomes produced by the method described in any one of claims 1 to 15 as an active ingredient.

17. The composition according to claim 16, characterized in that the composition is a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

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