Preparation method of stem cell-derived exosome through cell stretching and application of stem cell-derived exosome

Abstract

The present invention confirms that when stem cells are phase-changed into a gel (gel) state by mixing a hydrogel in a sol state composed of a polymer capable of sol-gel (gel) phase change with the stem cells, the stem cells can be cultured at a uniform high density in a 3D state. Furthermore, when the stem cells are cultured by the method, the stem cells are stretched by adjusting the hydrogel structure to transduce a physical stimulus to the cells, thereby enabling the activation of integrinin-FAK and catenin-Wnt signaling in the cells, and thus it is confirmed that the efficiency of exosome production is increased and functional components are increased. Furthermore, it is confirmed that in the exosome prepared by the method provided by the invention, miRNA components capable of regulating angiogenesis, tissue regeneration and inflammation are increased. Therefore, the exosome prepared by the method of the present invention can be effectively used as a composition for angiogenesis, tissue regeneration, or anti-inflammation.

Description

Technical Field

[0001] The present invention relates to a method for preparing functional exosomes derived from stem cells by increasing cell stretching and its use, and more specifically to a method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching and its use, the method comprising: mixing a hydrogel in a sol state and stem cells, wherein the hydrogel in the sol state has a ligand capable of binding to cellular integrins and is composed of polymer chains capable of undergoing a sol-gel phase transition; and phase transitioning the hydrogel to a gel state to prepare a 3D hydrogel in which stem cells are uniformly captured in three dimensions, and culturing the stem cells. Background Art

[0002] Stem cells have the characteristic of being anchorage-dependent, allowing them to survive and grow by attaching to a culture vessel. Exosomes are produced by stem cells and secreted into the culture medium. Exosomes contained in the culture medium can be isolated, concentrated, and purified to produce pharmaceuticals.

[0003] In order to prepare stem cell exosomes, it is necessary to provide a cell attachment matrix and culture fluid that stem cells can attach, survive and maintain function. In conventional monolayer culture (monolayer culture conditions, 2-dimensional culture conditions, 2D) environments, stem cell exosomes are prepared on an industrial scale, requiring a large number of culture containers and large-capacity culture fluids, and therefore have a problem of reduced efficiency. In order to overcome the low efficiency of the limited matrix area to which cells can attach in a 2D environment, it is necessary to prepare exosomes by high-density culture in a three-dimensional (3-dimensional, 3D) environment, thereby providing a matrix that can also attach many cells in a small area. Anchorage-dependent stem cells suffer from anoikis, that is, when cells are suspended in the absence of a cell attachment matrix, the cells are damaged and die. In order to carry out high-density culture so that the volume to which cells can attach can be increased and cell death can be suppressed, a 3D cell attachment matrix is ​​required. To achieve this purpose, a porous support, a hydrogel, a microsphere carrier, etc. are used as a 3D cell attachment matrix.

[0004] In the case of porous scaffolds, most cells adhere and grow along the surface of the support, forming a membrane along the surface. This prevents the exchange of oxygen, nutrients, and waste products to the interior of the support, resulting in central necrosis. In the case of microsphere carriers, while theoretically the area available for cell attachment is increased compared to 2D environments, the disadvantage is that during suspension culture, microspheres bind and aggregate, causing cell shedding from the microsphere surface, making long-term culture difficult.

[0005] Phase-change hydrogels are defined as hydrogels that readily mix with cells in a sol state before cross-linking and undergo a phase transition to a gel state after cross-linking. The hydrogel polymer solution in the solution state before cross-linking can be uniformly mixed with cells, allowing for a uniform 3D distribution of cells within the cross-linked hydrogel. Compared to porous supports or microsphere carriers, phase-change hydrogels have the advantage of having a well-developed interpenetrating network (IPN) between polymer chains, allowing for smooth movement of oxygen, nutrients, and metabolites.

[0006] However, it is known that hydrogels have weak biochemical properties and can be degraded by matrix metalloproteinases (MMPs) secreted from cells. If their physical properties are too hard, they inhibit cell binding and stretching, resulting in a decrease in the molecular biological functions of cells. In hydrogels, the interaction between cells and polymer chains is largely affected by the components that make up the polymer. In addition, the structural characteristics and physical properties of hydrogels vary depending on the concentration, molecular weight, and degree of cross-linking of the polymers that make up the hydrogel. Therefore, cell growth, migration, activity, stretching, and gene expression vary depending on the structure and physical properties of the hydrogel. As a result, the biological functions and activities of cells change, and the production rate of exosomes and the composition of genes within exosomes are also significantly affected.

[0007] By regulating the structure and physical properties of 3D hydrogels to enable them to function as cell attachment matrices capable of high-density cell culture in a 3D culture environment, and by optimizing the interactions between polymer chains within the hydrogels and cells, a technology is needed to generate exosomes from stem cells and exosomes with compositions that have enhanced biological functions. Summary of the Invention

[0008] Technical problem to be solved by the invention

[0009] Therefore, in the present invention, in order to efficiently produce stem cell-derived exosomes by adjusting the structure and physical properties of hydrogels, after intensive efforts, it was confirmed that a sol-state polymer solution composed of polymer chains with ligands that can bind to cellular integrins and capable of undergoing a sol-gel phase transition is mixed with stem cells. After a cross-linking reaction, the phase transition to a gel state occurs, allowing stem cells to be cultured uniformly at high 3D density within the 3D hydrogel. Using hydrogels to provide a 3D cell attachment matrix for stem cells can provide an environment where cells can be cultured at a density 10 to 100 times greater per area than in a 2D culture environment. This allows a large number of cells to be cultured using a small number of culture vessels, thereby improving the efficiency of exosome production.

[0010] Furthermore, a method is provided for controlling stem cell interactions within a 3D hydrogel by regulating the hydrogel's structural properties, including its physicochemical properties and pore size, to control gene expression and function. Depending on the hydrogel's properties, the cells' physiological activity, function, gene expression, exosome production rate, and exosome composition are altered.

[0011] The present invention provides a method for stretching cells by regulating the physicochemical and structural properties of a hydrogel. The method also induces activation of the integrin-FAK signaling pathway based on the degree of cell stretching, ultimately increasing the production rate of exosomes. Furthermore, the present invention provides a method for maximizing cell-cell bonding by regulating the hydrogel structure and activating intracellular gene expression by activating the β-catenin-Wnt signaling pathway, ultimately controlling the composition of miRNAs within exosomes produced in cells.

[0012] The present invention aims to provide a method for providing an industrial-scale exosome production medium by high-density culture of stem cells in 3D hydrogels. By stretching the cells, the production rate of exosomes can be increased, and a composition capable of enhancing the anti-inflammatory, tissue regeneration, and angiogenesis effects of the exosomes can be prepared.

[0013] Means for solving technical problems

[0014] To achieve the above objectives, the present invention provides a method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching, the method comprising: step (a), mixing a hydrogel in a sol state and stem cells, wherein the hydrogel in the sol state has a ligand capable of binding to the cell's integrin and is composed of polymer chains capable of undergoing a sol-gel phase transition; and step (b), phase-transforming the hydrogel into a gel state to prepare a 3D hydrogel in which the stem cells are uniformly captured in three dimensions and culturing the stem cells.

[0015] In a preferred embodiment of the present invention, the method can directly bind the ligand of the hydrogel to the cells without physical means to cause cell stretching.

[0016] In another preferred embodiment of the present invention, the hydrogel having a ligand capable of binding to the integrin can be a natural polymer material selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin, or a hydrogel copolymerized with a synthetic polymer material selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), and polyglycolic acid (PGA).

[0017] In another preferred embodiment of the present invention, in step (b), a cross-linking agent is added to the mixture of step (a) to enable the hydrogel to undergo a phase-transition to a gel state.

[0018] In another preferred embodiment of the present invention, in order to enhance the physicochemical properties of the 3D hydrogel in step (b), in step (a), at least one polymer material selected from the group consisting of polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), chitosan, gelatin, collagen, chondroitin, and hyaluronic acid may be further mixed. By enhancing the physical properties, "gel compaction" caused by the contractile force of cells causing the hydrogel to fall off from the culture container can be prevented.

[0019] In another preferred embodiment of the present invention, in order to enhance the stability by inhibiting the cellular degradation of the 3D hydrogel in step (b), in step (a), one or more matrix metalloproteinases (MMPs) selected from the group consisting of MMP-2, MMP-3, MMP-8 and MMP-9, and any one or more MMP inhibitors selected from the group consisting of marimastat, batimastat, ilomastat (GM6001) and cipemastat can be further mixed.

[0020] In another preferred embodiment of the present invention, in order to enhance the stability of the 3D hydrogel by inhibiting cellular degradation of the 3D hydrogel in step (b), in step (a), one or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid may be further mixed.

[0021] In another preferred embodiment of the present invention, the 3D hydrogel of step (b) has a porous structure with a size of 50 nm to 300 nm, provides cell-cell bonding, and has structural properties that promote cell stretching.

[0022] In another preferred embodiment of the present invention, the stem cells cultured in step (b) have a shape stretched in a three-dimensional axial plane, and the cells can be stretched to a length of 15 μm to 400 μm.

[0023] In another preferred embodiment of the present invention, the method can embed 1.0×10 4 ~5.0×10 6 Stem cells for culture.

[0024] In another preferred embodiment of the present invention, in step (b), the integrin-FAK-JNK-β-catenin signaling pathway can be activated in the stem cells cultured by regulating the physicochemical properties and structural characteristics of the hydrogel.

[0025] In another preferred embodiment of the present invention, in step (b), the Wnt signaling pathway in the cultured stem cells can be activated and the expression of Wnt signaling downstream target genes can be increased by regulating the physical properties and structural characteristics of the hydrogel.

[0026] In another preferred embodiment of the present invention, in the stem cell-derived exosomes prepared by the method, 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 may be increased.

[0027] In another preferred embodiment of the present invention, the stem cells may be adult stem cells or mesenchymal stem cells, preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, muscle-derived stem cells, myocardial-derived stem cells, peripheral nerve-derived stem cells, synovial-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

[0028] To achieve other purposes, the present invention provides a composition for angiogenesis, tissue regeneration, or anti-inflammation, comprising stem cell-derived exosomes prepared by the method as an active ingredient.

[0029] In a preferred embodiment of the present invention, the composition may be a pharmaceutical composition, a quasi-drug composition or a cosmetic composition.

[0030] Effects of the Invention

[0031] The present invention confirms that by mixing a sol-gel hydrogel composed of a polymer capable of undergoing a sol-gel phase transition with stem cells, when the phase transition is to a gel state, stem cells can be cultured at a uniform high density in a 3D state. In addition, when culturing stem cells by the method, the hydrogel structure is adjusted to stretch the stem cells to transduce physical stimulation to the cells, thereby achieving activation of intracellular integrin-FAK and catenin-Wnt signaling, thereby confirming that the generation efficiency of exosomes is increased and the functional components are increased. It has been confirmed that in the exosomes prepared by the method of the present invention, the miRNA components that can regulate angiogenesis, tissue regeneration, and inflammation are increased. Therefore, the exosomes prepared by the method of the present invention can be effectively used as angiogenesis, tissue regeneration, or anti-inflammatory compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1This photograph shows the cell characteristics of anchorage-dependent stem cells in a 2D culture environment. AT-MSCs are adipose-derived mesenchymal stem cells; BM-MSCs are bone marrow-derived mesenchymal stem cells; CB-MSCs are umbilical cord blood-derived mesenchymal stem cells; MyoCSCs are myocardial-derived stem cells; PNSCs are peripheral nerve-derived stem cells; SM-MSCs are muscle-derived stem cells; Syn-MSCs are synovial-derived mesenchymal stem cells; and UC-MSCs are umbilical cord-derived mesenchymal stem cells.

[0033] Figure 2 This is a flow cytometry histogram photograph showing the immunoexpression characteristics of cardiomyocyte-derived stem cells.

[0034] Figure 3 This image shows the differentiation characteristics of anchorage-dependent stem cells into adipocytes and osteoblasts. A T-MSCs are adipose-derived mesenchymal stem cells; BM-MSCs are bone marrow-derived mesenchymal stem cells; MyoC SCs are myocardium-derived stem cells; and PNSCs are peripheral nerve-derived stem cells.

[0035] Figure 4 Photos and results of (A) hydrogel porous structure and (B) pore size according to fibrinogen concentration.

[0036] Figure 5 Data showing (A) the extent of microfilament formation, which indicates the degree of cell stretching, and (B) the area of ​​microfilaments per cell within fibrin hydrogels were measured according to fibrinogen concentration.

[0037] Figure 6 These are photographs showing the expression of integrin-β1 and p-FAK by cardiomyocyte-derived stem cells within fibrin hydrogels according to fibrinogen concentration.

[0038] Figure 7 (A) Western blot images of p-FAK, p-JNK, and p-β-catenin expression in cardiomyocyte-derived stem cells within 3D fibrin hydrogels at varying fibrinogen concentrations. (B) p-FAK, p-JNK, and p-β-catenin expression levels in 2D culture, corrected for cardiomyocyte-derived stem cell expression levels. *, p < 0.05 compared to expression levels in 2D culture; **, p < 0.01 compared to expression levels in 2D culture.

[0039] Figure 8The results of Wnt signaling mRNA expression in cardiomyocyte-derived stem cells cultured in 3D fibrin hydrogels are corrected for mRNA expression in cardiomyocyte-derived stem cells cultured in 2D environments. (A) Expression rates of Wnt ligand-related mRNAs, (B) Wnt receptor-related mRNAs, and (C) downstream mRNAs of Wnt signaling pathway targets. *, p < 0.05 compared to 2D culture, **, p < 0.01 compared to 2D culture.

[0040] Figure 9 (A) Immunofluorescence staining of exosome markers CD9, CD63, and CD81 (red) and pFAK (green) was used to determine the production rate of exosomes from cardiomyocytes in 3D fibrin hydrogels according to fibrinogen concentration. (B) Quantification of immunofluorescence intensity. **, p < 0.01 compared to fibrin hydrogels prepared with 5 mg and 10 mg of fibrinogen.

[0041] Figure 10 To determine the production rate of exosomes from cardiomyocytes derived from stem cells in 3D fibrin hydrogels and 2D culture environments, depending on fibrinogen concentration, (A) shows the number of exosomes per ml measured using a nanoparticle tracking analyzer, and (B) shows the protein content. *, p < 0.01 compared to 2D culture.

[0042] Figure 11 Diameters of exosomes from cardiomyocytes were measured using a nanoparticle tracking analyzer in 3D fibrin hydrogels and 2D culture environments according to fibrinogen concentrations. (A) Mean diameter and (B) mode diameter of exosomes are shown.

[0043] Figure 12 This is a flow cytometry dot plot image used to analyze the immunogenicity of cardiomyocyte-derived stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments.

[0044] Figure 13This value represents the measured exosome production rate based on stem cell sources in 3D fibrin hydrogels 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, myocardium-derived stem cells, PNSC, peripheral nerve-derived stem cells, SM-MSC, muscle-derived stem cells, Syn-MSC, synovium-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.

[0045] Figure 14 This value measures the exosome production rate based on the seeding density of cardiomyocyte-derived stem cells within 3D fibrin hydrogels. *, p < 0.05 compared to 1.0E+04, **, p < 0.01 compared to 1.0E+04.

[0046] Figure 15 These values ​​are based on the production rates of (A) microfilaments and (B) exosomes by myocardial stem cells in a 3D culture environment using natural polymer components of hydrogels. *, p < 0.05 compared to gelatin, hyaluronic acid, and chondroitin; **, p < 0.01 compared to gelatin, hyaluronic acid, and chondroitin.

[0047] Figure 16 These are the measured values ​​for (A) microfilament formation and (B) exosome production by myocardial stem cells in synthetic polymer hydrogels and synthetic polymer and fibrin mixed hydrogels. *, p < 0.05 compared to synthetic polymer hydrogels; **, p < 0.01 compared to synthetic polymer hydrogels.

[0048] Figure 17 This figure shows the miRNA expression profiles of exosomes derived from cardiomyocytes cultured in 3D fibrin hydrogels and 2D cultures. The miRNA expression ratios are expressed as ratios normalized to those in bone marrow-derived mesenchymal stem cells (BM-MSCs). *, p < 0.05 compared to 2D culture; **, p < 0.01 compared to 2D culture.

[0049] Figure 18This chart compares the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to induce fibroblast growth. Comparisons were made using a statistic corrected for the number of cells in the group receiving the vehicle-free culture medium. AT-MSCs, adipose-derived mesenchymal stem cells, BM-MSCs, bone marrow-derived mesenchymal stem cells, MyoCSCs, myocardial-derived stem cells, PNSCs, peripheral nerve-derived stem cells, SM-MSCs, muscle-derived stem cells, *, p < 0.05 compared to 2D culture environment, **, p < 0.01 compared to 2D culture environment.

[0050] Figure 19 This chart compares the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to induce endothelial cell growth. Comparisons were made using a statistic corrected for the number of cells in the group receiving the vehicle-free culture medium. AT-MSCs, adipose-derived mesenchymal stem cells, BM-MSCs, bone marrow-derived mesenchymal stem cells, MyoCSCs, myocardial-derived stem cells, PNSCs, peripheral nerve-derived stem cells, SM-MSCs, muscle-derived stem cells. *, p < 0.05 compared to 2D culture environment, **, p < 0.01 compared to 2D culture environment.

[0051] Figure 20 This value measures the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to induce endothelial cell angiogenesis. AT-MSCs, adipose-derived mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; MyoCSCs, myocardial-derived stem cells; PNSCs, peripheral nerve-derived stem cells; SM-MSCs, muscle-derived stem cells. *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0052] Figure 21 These values ​​are the results of measuring the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to inhibit TNF-α secretion from inflammatory cells. AT-MSCs, adipose-derived mesenchymal stem cells, BM-MSCs, bone marrow-derived mesenchymal stem cells, MyoCSCs, myocardial-derived stem cells, PNSCs, peripheral nerve-derived stem cells, SM-MSCs, muscle-derived stem cells. *, p < 0.05 compared to 2D culture environment, **, p < 0.01 compared to 2D culture environment.

[0053] Figure 22These values ​​are the results of measuring the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to inhibit IL-1β secretion from inflammatory cells. AT-MSCs, adipose-derived mesenchymal stem cells, BM-MSCs, bone marrow-derived mesenchymal stem cells, MyoCSCs, myocardial-derived stem cells, PNSCs, peripheral nerve-derived stem cells, SM-MSCs, muscle-derived stem cells. *, p < 0.05 compared to 2D culture environment, **, p < 0.01 compared to 2D culture environment. DETAILED DESCRIPTION

[0054] Hereinafter, the present invention will be described in detail.

[0055] In one aspect, the present invention relates to a method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching, the method comprising: step (a), mixing a hydrogel in a sol state and stem cells, wherein the hydrogel in the sol state has a ligand capable of binding to cellular integrins and is composed of polymer chains capable of undergoing a sol-gel phase transition; and step (b), phase-transforming the hydrogel into a gel state to prepare a 3D hydrogel in which stem cells are uniformly captured in three dimensions, and culturing the stem cells.

[0056] More specifically, the present invention relates to a method comprising: step (a), mixing a hydrogel in a sol state and stem cells, wherein the hydrogel in the sol state has a ligand capable of binding to the integrin of the cells and is composed of polymer chains capable of undergoing a sol-gel phase transition; step (b), phase-changing the hydrogel into a gel state to prepare a 3D hydrogel in which the stem cells are uniformly captured in three dimensions and culturing the stem cells; step (c), stretching the cells through 3D binding between the cells and the polymer chains, transducing physical stimulation into the cells to enhance the integrin-FAK signaling pathway, thereby increasing the production rate of stem cell-derived exosomes; and step (d), enhancing the β-catenin-Wnt signaling pathway through cell-cell binding to increase the miRNA components in the stem cell-derived exosomes that act on anti-inflammatory, tissue regeneration, and angiogenesis.

[0057] In the present invention, the stem cells can be adult stem cells or mesenchymal stem cells, preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, myocardium-derived stem cells, muscle-derived stem cells, peripheral nerve-derived adult stem cells, synovial-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

[0058] In the present invention, the method may be characterized by binding the ligand of the hydrogel to the cells to stretch the cells without a physical device.

[0059] The present invention is characterized in that the hydrogel having a ligand capable of binding to the integrin can be a natural polymer material selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin, or can be a hydrogel copolymerized with the natural polymer material and a synthetic polymer material selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), and polyglycolic acid (PGA). Preferably, the hydrogel can be fibrinogen, collagen, or gelatin.

[0060] The present invention is characterized in that, in the step (b), a cross-linking agent may be added to the mixture of the step (a) to cause a phase transition to a gel state.

[0061] The polymer has a native ligand that can directly bind to cellular integrins and can therefore be used in the present invention.

[0062] Among these polymers, collagen, fibrin, gelatin, etc. can adjust the polymer content and cross-linking density, thereby adjusting the fine structure and physical properties within the hydrogel, and as a result, the production rate and composition of cellular exosomes can be adjusted.

[0063] On the contrary, the hydrogel has the characteristics of biodegradation and is easily decomposed by matrix metalloproteinases (MMPs) secreted by cells. In addition, weak physical properties also become a disadvantage because after the polymer chains bind to cells, the hydrogel will shrink due to cell-mediated contraction, resulting in the loss of its structural function as a 3D matrix. In order to overcome these biochemical and physical shortcomings, technologies that enhance biodegradation and structural properties are needed, and the stability of biodegradation can be improved by introducing MMP inhibitors or antifibrinolytic agents. In addition, the use of hydrogels whose physical properties are enhanced by copolymerizing with polymers such as PEG, PLGA, PGF, chitosan, hyaluronic acid, etc. that are not decomposed by MMP and have high physical strength can increase resistance to cell-mediated contraction.

[0064] Specifically, in order to adjust the physical strength of the 3D hydrogel in step (b), in step (a), 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.

[0065] In addition, in order to enhance the stability by inhibiting the cell degradation of the 3D hydrogel in step (b), in step (a), 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 mixed.

[0066] The present invention is characterized in that the 3D hydrogel of step (b) has a porous structure with a size of 50 nm to 300 nm, thereby supporting cell-cell binding and having structural properties that can promote cell stretching.

[0067] The present invention is characterized in that the stem cells cultured in step (b) can have a shape stretched in a 3D axial plane, and the cells can be stretched to a length of 15 μm to 400 μm. In addition, the method can embed 1.0×10 cells per ml of hydrogel at a high density. 4 ~5.0×10 6 Stem cells for culture.

[0068] Hydrogels can provide a 3D cell attachment matrix to which cells can attach at high density. Therefore, the present invention can provide a method for increasing the efficiency of exosome production by high-density culture at a higher density than conventional two-dimensional (2D) culture. For lipid-dependent cells such as mesenchymal stem cells, it is impossible to culture 100 cells per cm in a 2D environment. 2 5.0×10 4 Cell density is above 1000, whereas in 3D environment, cells can be cultured per cm 3 0.5~5.0×10 6 Therefore, compared with 2D, a cell attachment matrix can be provided that can culture cells at a density 10 to 100 times higher.

[0069] The rate of exosome production is proportional to the number of cells that produce exosomes. In particular, for stem cells that are anchorage-dependent cells, the area of ​​the matrix that can be attached is an absolute variable to determine the number of cells that can be cultured, and the exosome yield is determined based on the number of cells cultured. The present invention provides a technology that uses hydrogels to attach cells to a 3D matrix at a high density, thereby providing a technology that can generate large-scale levels of exosomes even when using a small-sized culture container. In particular, the advantage of using a sol-gel phase transition hydrogel is that it mixes with cells in a sol state and changes phase to a gel state, so that stem cells can be uniformly embedded in the 3D hydrogel. When culturing stem cells in a T175 flask, it is possible to 2 The maximum number of stem cells cultured in a flask is 7 to 8E+06, but the number of cells that can be cultured in a 3D hydrogel with a thickness of 1 cm can reach 8E+08.

[0070] Specifically, the present invention can regulate the production rate of stem cell exosomes and their ability to induce inflammation, tissue regeneration, and angiogenesis by adjusting the structure and physicochemical properties of the hydrogel. A method is provided in which cells bind to polymer chains that constitute the nanostructure within the hydrogel, inducing cell stretching. As a result, compared to a 2D environment, physical stimulation activates integrins, FAK, β-catenin, and Wnt signaling. A method is provided in which, by adjusting the structure and physicochemical properties of the hydrogel, cell function is modulated, ultimately increasing the efficiency of stem cell exosome production and enhancing functional components. Sparse nanostructures within the hydrogel increase stem cell stretching and intercellular binding, while dense nanostructures reduce stem cell stretching and intercellular binding. Depending on the degree of cell stretching and intercellular binding, the activity levels of the FAK, integrin, β-catenin, and Wnt signaling pathways vary, affecting the production rate of stem cell exosomes and the composition of functional components within the exosomes.

[0071] In one specific embodiment of the present invention, a hydrogel is prepared using fibrinogen. Specifically, stem cell-derived exosomes can be prepared by a method comprising the following steps, the method comprising: step (1), mixing a sol-state hydrogel polymer solution and stem cells; and step (2), adding a cross-linking agent to the mixture to prepare a gel-state hydrogel in which stem cells are uniformly captured in 3D and culturing the stem cells.

[0072] Furthermore, the structural characteristics and physicochemical properties of hydrogels can be adjusted based on the polymer content and degree of cross-linking. Higher polymer content results in a more robust structure, while lower content allows for a sparser structure. However, the addition of MMP inhibitors, antifibrinolytic agents, and synthetic polymers can mitigate the shortcomings of sparsely structured hydrogels, enhance their stability against degradation, and mitigate the potential for hydrogel compaction and shedding from culture vessels due to weak physical strength.

[0073] In another specific example of the present invention, the structural characteristics and physical properties of fibrin hydrogel can be adjusted by adjusting the fibrinogen concentration. It has been confirmed that as the fibrinogen concentration increases, the pore size decreases, resulting in a narrower pore spacing, and a dense and solid structure can be prepared. The lower the fibrinogen content, the more sparse the hydrogel structure can be prepared.

[0074] That is, the present invention confirmed that when a hydrogel is prepared with a fibrinogen 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 a porous structure with a size of 100 nm to 300 nm, and the stem cells in the fibrin hydrogel composed of low concentration fibrinogen are stretched to a length of 15 μm to 400 μm, and the cell-cell bonding is increased ( Figure 4 and Figure 5 ).

[0075] In addition, when the fibrin hydrogel prepared in the present invention was used to culture stem cells, it was confirmed that the stretching of the stem cells was proportional to the degree of cell-cell bonding in the sparsely structured fibrin hydrogel, and the FAK, integrin, Wnt, and β-catenin cell signaling pathways were activated ( Figures 6 to 9 ), the production of exosomes increased ( Figure 10 and Figure 11 ).

[0076] Furthermore, when comparing the exosomes prepared in the 3D hydrogel culture environment of the present invention with the exosomes prepared in 2D culture, it was confirmed that there was no significant difference in the size and immune expression characteristics of the exosomes ( Figure 12 ), but based on the production of stem cell-derived exosomes ( Figure 13 ), angiogenesis, tissue regeneration, and anti-inflammatory regulatory miRNA expression levels increased significantly ( Figure 17 ). In addition, the exosomes prepared in the 3D hydrogel culture environment of the present invention were confirmed to have significantly increased angiogenesis, tissue regeneration, and inflammation regulation effects compared to the exosomes prepared in 2D culture ( Figures 18 to 22 ).

[0077] The present invention confirms that the stretchability of embedded stem cells can be regulated by adjusting the nanostructure of the hydrogel. As cell stretch increases, physical stimulation activates stem cell signaling pathways, increasing exosome production. Furthermore, the exosomes prepared using the present method have been shown to increase the types of cell growth, wound repair, angiogenesis, and inflammation-regulating proteins and miRNAs expressed within the exosomes, thereby enhancing the angiogenic, tissue regeneration, and anti-inflammatory effects of the exosomes.

[0078] In another aspect, the present invention relates to a composition for angiogenesis, tissue regeneration, or anti-inflammation, comprising the stem cell-derived exosomes prepared by the method as an effective ingredient.

[0079] In the present invention, the composition may be a pharmaceutical composition, a quasi-drug composition or a cosmetic composition.

[0080] The angiogenic composition can induce or increase the migration of vascular endothelial cells and can promote angiogenesis in diseases requiring angiogenesis.

[0081] The disease requiring angiogenesis may be any one or more selected from the group consisting of wounds, burns, ulcers, necrosis, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease, and alopecia.

[0082] The composition for tissue regeneration can be used to regenerate tissue selected from, but not limited to, skin, cartilage, bone, blood vessels, brain, liver, heart, ligaments, muscle, spinal cord, blood, bone marrow, lungs, teeth, nerves, cornea, retina, esophagus, spine, kidney, pancreas, or urethra. Furthermore, the composition for tissue regeneration can be used to improve skin condition, fill wrinkles, or sculpt facial or body contours. Furthermore, it can be effectively used as a dermal filler.

[0083] The anti-inflammatory composition can be a pharmaceutical composition for preventing or treating an inflammatory disease, and the inflammatory disease can be any one 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.

[0084] In the present invention, the inflammatory disease can be any one selected from the group consisting of atopy, psoriasis, dermatitis, allergy, arthritis, rhinitis, otitis media, pharyngitis, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, inflammatory bowel disease, ankylosing spondylitis and systemic lupus erythematosus (SLE), atherosclerosis, asthma, arteriosclerosis, edema, rheumatoid arthritis, delayed hypersensitivity (type IV hypersensitivity), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, arthritis, cystic fibrosis, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, vascular restenosis, glomerulonephritis and gastrointestinal allergy.

[0085] The pharmaceutical compositions of the present invention can be formulated and used in various dosage forms according to conventional methods. For example, they can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups. They can also be formulated into external preparations, suppositories, and sterile injections. Each dosage form may also include a pharmaceutically acceptable carrier, excipient, and diluent. In addition, according to conventional methods, they can be formulated into external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injections.

[0086] Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When the pharmaceutical composition is formulated or formulated, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0087] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., into the composition. In addition, in addition to simple excipients, lubricants such as magnesium styrene talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., which, in addition to water and liquid paraffin as commonly used simple diluents, may include various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solutions, suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As the base of the suppository, semi-synthetic Witepsol, macrogol, Tween 61, cocoa butter, laurel butter, glycerin gelatin and the like can be used.

[0088] As used herein, the term "administering" refers to providing a pharmaceutical composition of the present invention to an individual by any appropriate method. The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount of the active ingredient or pharmaceutical composition that is determined by the researcher, veterinarian, physician, or other clinician to induce a biological or medical response in a tissue system, animal, or human, i.e., in an amount that is effective to induce symptomatic relief of the disease or disorder being treated. The therapeutically effective dose and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect, as will be apparent to one of ordinary skill in the art. Therefore, one of ordinary skill in the art can readily determine the optimal dose to be administered, which can be adjusted based on various factors, including the type of disease, the severity of the disease, the amount of active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the rate of secretion of the composition, the duration of treatment, and concomitant medications. The pharmaceutical composition of the present invention can be administered to an individual by various routes. For example, administration can be performed intravenously, intraperitoneally, intramuscularly, intraarterially, orally, intracardially, intramedullary, intrathecally, transdermally, enterally, subcutaneously, sublingually, or topically, but is not limited thereto. The pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg / kg / day, and can also be administered once a day or in divided doses.

[0089] As used herein, the term "quasi-drug" refers to an article used for the purpose of diagnosing, treating, ameliorating, alleviating, curing, or preventing human or animal diseases that has milder effects than pharmaceuticals. For example, under the Pharmaceutical Affairs Act, quasi-drugs include products used to treat or prevent human and animal diseases, as well as products that have minimal or no direct effects on the human body, excluding those used as pharmaceuticals.

[0090] The quasi-drug composition of the present invention is not particularly limited in its dosage form. For example, it can be a cosmetic composition having the dosage form of a skin softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a facial mask, a facial mask sheet, a gel, or a skin adhesive cosmetic. In addition, it can also be a transdermal dosage form such as a lotion, an ointment, a gel, a cream, a patch, or a spray.

[0091] Furthermore, within each dosage form, quasi-drug compositions can be formulated with other ingredients arbitrarily selected based on the dosage form or intended use of other quasi-drugs. The amount of active ingredient incorporated can be appropriately determined based on the intended use (inhibition or relief). For example, conventional adjuvants and carriers such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances may be included.

[0092] 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-based, lotion-based, wax-based or spray, but is not limited thereto.

[0093] In addition, the cosmetic composition of the present invention may further comprise adjuvants conventionally used in the field of cosmetics or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion chelating agents and chelating agents, preservatives, vitamins, occlusive agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients conventionally used in cosmetics. Furthermore, the above ingredients can be incorporated in amounts commonly used in the field of dermatology.

[0094] These embodiments are only used to illustrate the present invention, and the scope of the present invention should not be interpreted as being limited to these embodiments. This is obvious to those skilled in the art to which the present invention belongs.

[0095] Example 1: Stem cell isolation and culture

[0096] In the present invention, various stem cells were isolated and cultured to prepare stem cell-derived exosomes.

[0097] Adipose-derived mesenchymal stem cells (AT-MSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), skeletal muscle-derived mesenchymal stem cells (SM-MSCs), myocardium-derived stem cells (MyoCSCs), peripheral nerve-derived stem cells (PNSCs), synovium-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.

[0098] Plasma-derived fibrinogen was dissolved in phosphate-buffered saline (PBS) 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. A scalpel was used to cut the adipose tissue (AT), bone marrow (BM), myocardium (myocardium), skeletal muscle (SM), synovium, peripheral nerve (peripheral nerve), and umbilical cord (UC) tissues to be embedded in the hydrogel into 0.2 to 2 mm pieces.3 After slicing the tissue slices to the required size, remove the hematoma and fibrous tissue from the tissue slices before taking them out. Wash the tissue slices 3 times with PBS to remove the blood cells in the tissue. After washing, mix each 10㎎ tissue slice with 1ml of thrombin solution and place it in an ice bucket for 10 minutes. Then, add an equal amount of fibrinogen solution, mix well, transfer the mixture to a culture container, and place it in a 37°C incubator for 1 hour to form a gel. The slices to which the fibrinogen solution was added are embedded in a fibrin hydrogel composed of 0.25% fibrinogen and 0.5unit / ml of thrombin, which is the final wound repair matrix.

[0099] The composition of the culture medium used for organ culture is as follows: 45% (vol / vol) Dulbecco's modified Eagle's medium (DMEM), 45% Ham's F12, 10% fetal bovine serum (FBS, Invitrogen), 20 ng / ml EGF, 2 ng / ml bFGF, 10 ng / ml IGF, 10 μg / ml gentamicin (Invitrogen), and 200 mg / ml tranexamic acid. Add twice the volume of organ culture medium to the gel volume. After adding the organ culture medium, place the culture container on an orbital shaker and gently stir at 30 rpm for 14 days. Replace the organ culture medium with fresh one twice a week.

[0100] After culturing the organ for 2 weeks, the culture medium was removed, and DMEM was added, and the organ was washed three times, each time for 10 minutes, to remove the organ culture medium remaining in the tissue slices and wound repair matrix.

[0101] After adding fresh organ culture medium without PAI (plasminogen activator inhibitor), place the culture container on a gyratory shaker and stir at 30 rpm for 1 hour at 37°C to allow the stem cells that have migrated and grown in the wound repair matrix to be released into the culture medium. The stem cells that have been released into the culture medium are collected, transferred to a 50 ml centrifuge tube, and centrifuged at 300 × g for 10 minutes. After removing the supernatant, disperse the cells into 10 ml of cell culture medium and count the cells using a hemocytometer. The number of cells is 3000 / cm 2 The cells are seeded into culture vessels at a high density and the stem cells are expanded and cultured in a monolayer culture environment.

[0102] Umbilical cord blood was centrifuged through a Ficoll density gradient to isolate mononuclear cells. The process of adding PBS and centrifuging was repeated three times. After washing, the cells were suspended in cell culture medium, seeded, and cultured for two weeks. Colonies formed after culture were isolated by trypsinization and expanded using conventional monolayer culture methods. All stem cells used for exosome production were harvested during four subcultures.

[0103] like Figure 1 As shown, cells isolated and cultured from adipose tissue, bone marrow, umbilical cord blood, myocardium, skeletal muscle, peripheral nerve, synovium, and umbilical cord all exhibited anchorage-dependent growth characteristics by attaching to the vessel in which they were grown, and typical spindle-shaped stromal cell characteristics were confirmed.

[0104] Example 2: Analysis of the immune expression characteristics of stem cells

[0105] After organ culture, the immune expression characteristics of stem cells expanded using conventional monolayer culture were analyzed. One hundred thousand cells were reacted with fluorescently labeled anti-human antibodies targeting CD31, CD34, CD44, CD45, CD73, and CD105. The positive rate of each antibody was analyzed using a FACSCalibur (Becton Dickinson, USA) flow cytometer (FCM), and the expression rate was analyzed in a sample of more than 10,000 cells.

[0106] Table 1

[0107] Stem cell marker expression rate (%)

[0108]

[0109] like Figure 2 As shown in Table 1, more than 90% of anchorage-dependent cells isolated and cultured from adult tissues expressed CD44, CD73, and CD105, markers of stromal cells. However, expression of CD31, CD34, and CD45, markers of vascular endothelial cells, hematopoietic cells, and immune cells, was less than 2%, indicating that the cells used to produce exosomes had a purity of more than 90% stromal cells.

[0110] Example 3: Confirmation of Adipocyte and Osteoblast Differentiation Characteristics of Stem Cells

[0111] To evaluate the ability of stem cells to differentiate into adipocytes, 200,000 cells were seeded into 24-well tissue culture plates and cultured for 14 days in 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 μg / ml insulin (Sigma). Cells were then stained with 0.5% Sudan Black (Sigma), an indicator of intracellular fat accumulation, for one hour at room temperature to determine whether the cells were differentiating into adipocytes based on the accumulation of fat in the cytoplasm.

[0112] To evaluate the ability of stem cells to differentiate into osteoblasts, 200,000 cells were seeded into 24-well tissue culture plates and cultured for 14 days in 90% α-MEM supplemented with 10% CS, 50 μg / ml ascorbic acid, 0.1 μM dexamethasone (Sigma), and 1 mM calcium glycerophosphate (Sigma). Mineral accumulation was stained with 1 μM calcein, and differentiation of stem cells into osteoblasts was assessed using fluorescence microscopy.

[0113] like Figure 3 The results show that anchorage-dependent stem cells differentiate into adipocytes and osteoblasts. Adipocyte differentiation results in the accumulation of multiple vesicles in the cytoplasm, which are Sudan Black positive. This accumulation of fat vesicles in the cytoplasm confirms the differentiation into adipocytes.

[0114] Regarding differentiation into osteoblasts, mineral crystals were observed in the generated extracellular matrix using phase contrast microscopy, and these minerals were confirmed to be calcium phosphate using calcein fluorescence microscopy.

[0115] The degree of differentiation into adipocytes and osteoblasts varies depending on the tissue of origin, but among all stem cells, stem cells with multipotential differentiation have been confirmed along with the confirmation of differentiation into adipocytes and osteoblasts.

[0116] Example 4: Hydrogel structure regulation based on polymer concentration

[0117] After dissolving fibrinogen (Sigma) in DMEM, four fibrinogen solutions with concentrations of 2.5, 5.0, 10.0, and 20.0 mg / ml were prepared. Aminomethylbenzoic acid (Sigma) was added to the fibrinogen solution at a concentration of 100 μg / ml. Thrombin (Sigma) was dissolved in DMEM to prepare a 1 unit / ml thrombin solution. To confirm the size and connectivity of fibrin pores, Alexa Fluor 546-conugated fibrinogen (Molecular Probes) was mixed at a ratio of 1:10 to prepare a fibrinogen solution. This solution was then mixed with a 1 unit / ml thrombin solution at a ratio of 1:1, and then a fibrin hydrogel was prepared by polymerization and cross-linking at 37°C for 1 hour. Confocal microscopy (Zeiss LSM 900) was used to evaluate the structural properties of fibrin hydrogels generated at four different fibrinogen concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml).

[0118] like Figure 4 As shown in FIG, as the fibrinogen concentration of the constituent polymer of the prepared fibrin hydrogel increases, the size of the pores decreases, forming a hydrogel with a dense porous structure.

[0119] On the contrary, as the fibrinogen concentration decreases, the size of the pores decreases, forming a sparsely structured hydrogel. Figure 4 As shown in Figure 3B, the pore sizes of the fibrin hydrogels prepared with fibrinogen solutions of four different concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) were 265.7, 165.2, 76.8, and 38.5 nm, respectively, showing that as the fibrinogen concentration increased, the diameter of the pores decreased significantly and the structural characteristics were solid.

[0120] Example 5: Cell stretching based on hydrogel nanostructures

[0121] The interaction between hydrogels and myocardial-derived stem cells (MyoCSCs) was evaluated by 3D culture of stem cells based on their physical and structural properties.

[0122] 1 ml of fibrinogen solution at four different concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) was mixed with 1 million MyoCSCs and then mixed with the same volume of thrombin solution. Subsequently, a polymerization and cross-linking reaction was performed at 37°C for 1 hour to prepare a fibrin hydrogel embedded with MyoCSCs. A serum-free culture medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cysteine ​​in DMEM / F12 was prepared as a cell culture solution. This solution was added to the fibrin hydrogel embedded with MyoCSCs and cultured for 12 hours.

[0123] After incubation, the stem cell-embedded hydrogels were fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, the cells were reacted with Alexa Fluor 488-labeled phalloidin (Invitrogen) for 60 minutes. Confocal microscopy was then used to measure the length of microfilaments formed in the cytoplasm and assess the degree of cell stretching.

[0124] like Figure 5 As shown, the degree of cell stretching varies depending on the type of fibrin hydrogel, along with the microfilaments in the cytoplasm. Stem cells in the hydrogel differ in shape from those in 2D culture environments; stretching of the cells can be confirmed in the 3D axial plane, and the cytoplasm exhibits long, thin characteristics.

[0125] The degree of cell stretching of stem cells in sparse fibrin hydrogels formed with low fibrinogen concentrations was higher, while for hydrogels formed with high fibrinogen concentrations, cell stretching was significantly reduced. Figure 5 As shown in Figure 2, the cell stretching length was the highest on the sparsely structured fibrin hydrogel. The cell stretching lengths of stem cells in fibrin hydrogels prepared with four fibrinogen concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) were 112.5, 75.0, 25.2, and 7.4 μm, respectively. Remarkably high microfilament lengths were confirmed in the sparsely structured hydrogel, and it was confirmed that physical cell stretching can be adjusted according to the structural and physical properties of the hydrogel.

[0126] Example 6: Activity of stem cell signaling based on hydrogel nanostructures

[0127] The interaction between hydrogels and myocardial-derived stem cells (MyoCSCs) was evaluated by culturing them in 3D.

[0128] After preparing a hydrogel embedded with stem cells using the same method as in Example 5, the cells were fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, the cells were reacted with primary antibodies against β-integrin (Cell Signaling Technology, USA) and p-FAK (Cell Signaling Technology, USA) at 37°C for 1 hour, followed by reaction with Alexa Fluor 488-conugated anti-mouse IgG (Invitrogen, USA) for 30 minutes.

[0129] Cell nuclei were stained with DAPI (diamidino-2-phenylindole, Molecular Probes), and the expression of β-integrin and p-FAK was assessed using confocal microscopy. Western blotting was performed to evaluate integrin-mediated cell transduction pathways. After extracting intracellular proteins within 3D hydrogels or 2D culture environments, polyacrylamide gel electrophoresis (PAGE) was performed. PAGE gels were blotted onto signal enhancer Immobilon membranes (Sigma) and incubated with primary antibodies against p-FAK (Cell Signaling Technology, Inc., USA), p-JNK (Cell Signaling Technology, Inc., USA), and p-β-catenin (Cell Signaling Technology, Inc., USA) for 1 hour, followed by incubation with AP-conjugated secondary antibodies for 30 minutes. The band intensity was then measured after reaction with ECL substrate (Thermo Fisher Scientific, USA). The results were compared and normalized using the expression intensity of β-actin.

[0130] like Figure 6 As shown, differences in the expression of β-integrin, p-FAK, and β-catenin were confirmed in stem cells embedded in 3D environments, depending on the hydrogel structural characteristics. Compared to 2D culture environments, 3D culture environments showed significantly increased expression of p-FAK, p-JNK, and β-catenin. In particular, since expression of p-FAK, p-JNK, and β-catenin increases in sparse hydrogel structures, the expression rates are inversely proportional to the concentration of the hydrogel's constituent polymers.

[0131] Compared to 2D culture, the expression of P-FAK in hydrogels containing fibrinogen at concentrations of 1.25, 2.5, 5.0, and 10.0 mg increased by 85.4, 67.8, 34.2, and 0.7 times, respectively. Compared to 2D culture, the expression of P-JNK in hydrogels containing fibrinogen at concentrations of 1.25, 2.5, 5.0, and 10.0 mg increased by 35.4, 25.4, 12.6, and 0.8 times, respectively. The expression of p-β-catenin increased by 12.5, 9.4, 3.5, and 0.9 times, respectively.

[0132] The above results confirm that with the degree of cell stretching, microfilament formation increases, and at the same time, the expression of p-FAK, pJ NK, and β-catenin also increases, thus confirming that cell stretching can activate cell signaling pathways.

[0133] Example 7: Expression of Wnt signaling pathway messenger RNA (mRNA) in stem cells in 2D and 3D culture environments characteristic

[0134] In the present invention, a polymerase chain reaction (PCR) microarray kit (RT 2 Profiler PC RArray, Qiagen, Germany) was used to evaluate the mRNA expression rate of the Wnt signaling pathway in stem cells cultured from 2D to 3D hydrogel culture environments.

[0135] Total RNA was extracted from MyoCSCs cultured in a 2D environment using TRIzol (Invitrogen), and then complementary deoxyribonucleic acid (cDNA) was synthesized using the reverse transcriptase provided in the kit.

[0136] 3D hydrogel was prepared with 1.25㎎ / ml fibrinogen and 0.5unit / ml thrombin solution. When preparing the hydrogel, 1.0×10 6 MyoCSCs were cultured for 3 days, and total RNA and cDNA were synthesized using the same method as in the 2D culture environment.

[0137] The expression rates of p-FAK, p-JNK, and p-β-catenin were confirmed. 2 The expression of 82 genes, including Wnt ligands, receptors, and downstream target genes, was profiled in the Profiler PCR Array (Qiagen, Germany). Expression ratios were corrected for housekeeping genes and compared with MyoCSCs in 2D culture using the ΔΔCT method, and expressed as expression ratios.

[0138] like Figure 8 As shown, in a 3D hydrogel environment, the expression of Wnt ligands, receptors, and Wnt target downstream mRNAs increased significantly. The Wnt signaling pathway is activated by extracellular physical stimulation, and in particular, the Wnt signaling pathway increases with the degree of microfilament formation of actin filaments in the cytoplasm.

[0139] As described in this example, physical stimulation of stem cells is induced in a 3D hydrogel culture environment, resulting in the promotion of actin filament organization and cell stretching, which leads to a significant increase in microfilament formation, which can be confirmed at the mRNA level, and ultimately the activation of the Wnt signaling pathway.

[0140] Compared with 2D culture, Wnt ligand mRNA expression increased 56.6-fold in WNT1, 32.3-fold in WNT10A, 15.4-fold in WNT10B, 17.0-fold in WNT11, 77.6-fold in WNT2B, 27.5-fold in WNT3A, and 47.0-fold in WNT 6 (p<0.05). Wnt receptor mRNA expression also increased in stem cells cultured in 3D.

[0141] Compared to 2D culture, 3D stem cell mRNA expression increased by 16.4-fold for FZD1, 57.9-fold for FZD10, 11.0-fold for FZD3, 7.3-fold for FZD5, 14.0-fold for FZD9, and 71.7-fold for SFRP2. Significant changes were also observed in the expression of downstream target genes affected by the Wnt signaling pathway.

[0142] Compared to 2D culture, stem cells cultured in 3D environments showed a 12.2-fold increase in CCND1, a 7.5-fold increase in CCND2, a 3.0-fold increase in CD44, a 90.2-fold increase in CDX1, a 63.1-fold increase in CDX2, a 47.7-fold increase in FGF4, a 62.5-fold increase in FGF9, a 6.7-fold increase in FN1, a 29.8-fold increase in MYC, a 45.0-fold increase in MYCN, a 49.3-fold increase in NANOG, an 84.1-fold increase in SOX2, and an 87.9-fold increase in VEGFA. These results suggest that modulating the 3D hydrogel structure to activate the Wnt signaling pathway in stem cells can lead to increased expression of functional genes.

[0143] Example 8: Generation of exosomes in stem cells based on 3D hydrogel nanostructures and cell stretching

[0144] The correlation between cell stretching, p-FAK, and exosome production of stem cells according to the structural properties of hydrogels was evaluated.

[0145] After preparing a stem cell-embedded hydrogel using the same method as described in Example 5, the cells were fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, the cells were incubated with the primary antibody p-FAK (Cell Signaling Technology, USA) at 37°C for 1 hour, followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen, USA) for 30 minutes. Exosome markers anti-CD9, CD63, and CD81 were incubated with markers (Thermo Fisher Scientific, USA) for 1 hour, followed by incubation with Alexa Fluor 594 (Molecular Probes, USA), and cell nuclei were stained with DAPI (diamidino-2-phenylindole, Molecular Probes, USA). The expression of p-FAK and the rate of exosome production in the cytoplasm were assessed by confocal microscopy.

[0146] Table 2

[0147] Intracellular exosome formation according to polymer (fibrinogen) concentration

[0148]

[0149] like Figure 9 As shown, the level of p-FAK expression in stem cells within 3D hydrogels increases proportionally to exosome production. p-FAK expression increases in direct proportion to cell stretching, and the number of cytoplasmic particles positive for exosome markers CD9, CD63, and CD81 increases significantly in accordance with the level of p-FAK expression. As previously shown, the intensity of exosome-positive particles in cells within sparse hydrogel structures prepared with low fibrinogen concentrations significantly increases. These results confirm that hydrogel structure can regulate p-FAK expression, and consequently, stem cell exosome production.

[0150] Example 9: Exosome secretion into cell culture medium based on 3D hydrogel nanostructures

[0151] The correlation between cell stretchability and the rate of exosome secretion into cell culture medium of cardiomyocyte-derived stem cells according to the structural properties of hydrogels was evaluated.

[0152] 1 ml of fibrinogen solution at four different concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) was mixed with 1 million MyoCSCs and then mixed with an equal volume of thrombin solution. The cells were then polymerized and cross-linked for 1 hour at 37°C to create a fibrin hydrogel embedded with MyoCSCs. A serum-free culture medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cysteine ​​in DMEM / F12 was prepared using cell culture solution and added to the fibrin hydrogel embedded with MyoCSCs, and the cells were cultured for 24 hours.

[0153] Exosomes were isolated from the cell culture fluid collected 24 hours later using tangential flow filtration (TFF) using a 300 kDa molecular weight cut-off cartridge (Repligen, USA). The concentrated and purified exosomes were analyzed using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical, UK) to assess the number of nanoparticles in the culture fluid. Protein content within the exosomes was determined using microBCA (Thermo Fisher Scientific, USA). Exosomes were isolated from the cell culture fluid obtained after culturing the same MyoCSCs in a 2D culture environment for 24 hours.

[0154] Table 3

[0155] Exosome production in 3D culture environments according to the polymer content of 3D hydrogel components

[0156]

[0157] like Figure 10 As shown in the results, it was confirmed that the number of exosomes produced and secreted from stem cells differed significantly depending on the structural characteristics of the hydrogel. The number of exosomes secreted into the cell culture medium was the highest in stem cells cultured in sparsely structured hydrogels prepared at low polymer concentrations. The number of exosomes secreted into the cell culture medium in fibrin hydrogels prepared at four fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml was 25.4 × 10 9 / ml, 16.1×10 9 / ml, 5.7×10 9 / ml, 1.1×10 9 / ml, and the number of exosomes was significantly higher in the sparsely structured fibrin hydrogel prepared with low fibrinogen concentration.

[0158] Similar to the number of exosomes, protein content was also found in the sparsely structured hydrogels, with significantly higher levels of protein secreted by stem cells. In fibrin hydrogels prepared at fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml, protein contents were 318.5, 213.1, 68.4, and 18.5 μg / ml, respectively. The sparsely structured fibrin hydrogels showed significantly higher levels of protein produced by stem cells and released into the cell culture medium. It was confirmed that, with the exception of the fibrin hydrogel concentration of 10.0 mg / ml, stem cells cultured in fibrin hydrogels can support significantly higher exosome secretion than stem cells cultured in 2D culture environments.

[0159] Example 10: Confirmation of the size of exosomes produced in 2D culture and 3D hydrogel culture environments

[0160] In the present invention, structural differences in exosomes produced according to culture environments were examined.

[0161] According to the method proposed in Example 9, myocardial stem cells were embedded in a 3D fibrin hydrogel and cultured, and then exosomes were isolated.

[0162] The size of nanoparticles in the culture medium of the concentrated and purified exosomes was measured using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical, UK). Exosomes were isolated from the culture medium obtained after culturing the same MyoCSCs in a 2D culture for 24 hours, and the nanoparticle size was measured using the same method.

[0163] like Figure 11 As shown, no differences in exosome particle size were observed depending on the culture environment. The average diameters of exosomes generated from gels containing fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml were 118, 116, 117, and 121 nm, respectively, which did not differ from the average diameter of 121 nm for exosomes generated in a 2D environment. The mode value, the most frequent nanoparticle size, also showed no difference between 2D and 3D culture environments, nor was any difference observed depending on the polymer concentration of the hydrogel composition.

[0164] Example 11: Immune expression characteristics of exosomes produced and secreted by stem cells in 2D culture and 3D hydrogel culture environments sex

[0165] The immunohistochemical properties of exosomes generated and isolated from cardiomyocyte-derived stem cells were analyzed using a multiplex bead kit (MACSPlex Exosome Kit, Miltenyi Biotec, Germany) in 2D and 3D culture environments.

[0166] According to the method proposed in Example 9, myocardial stem cells were embedded in 3D fibrin hydrogels for culture, and then exosomes were isolated.

[0167] After centrifugation at 2500 × g for 15 minutes, the supernatant was collected for analysis. 30 μl of exosomes were mixed with MACSPlex buffer at 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 incubation at room temperature for 30 minutes, the exosomes were analyzed for immunohistochemistry using flow cytometry, and the extent of expression was analyzed using median APC-A expression intensity.

[0168] Table 4

[0169] Immune expression characteristics of cardiomyocyte-derived stem cell exosomes according to culture environment

[0170]

[0171]

[0172]

[0173] As shown in Table 4 and Figure 12 As shown, there were no differences in the immune expression characteristics of exosomes produced by culture environment. The expression intensities of the exosomal markers CD9, CD63, and CD81 were high in exosomes produced in both 2D and 3D culture environments, but no differences were observed depending on the culture environment. Expression of the cardiomyocyte-derived stem cell markers CD105, CD44, and CD29 also showed no differences between 2D and 3D culture environments, with high expression in all exosomes.

[0174] Example 12: Exosome production rates according to stem cell types in 2D and 3D culture environments

[0175] In this study, we analyzed the production of exosomes based on the type of stem cells and the culture environment. We cultured adipose, bone marrow, umbilical cord blood, synovial, umbilical cord-derived mesenchymal stem cells, peripheral nerve-derived adult stem cells, and myocardial-derived stem cells in 2D and 3D environments.

[0176] The stem cells were embedded in a 3D fibrin hydrogel and cultured according to the method described in Example 9, and then exosomes were isolated. The exosome production rate of the concentrated and purified exosomes was measured using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical, UK).

[0177] like Figure 13 As shown, significant differences in the amount of exosomes produced were observed depending on the culture environment. Compared to 2D culture, the amount of exosomes produced in 3D culture increased significantly by 1.7 to 2.9 times. Significant increases in myocardial stem cells (MyoCSCs), peripheral neural stem cells (PNSCs), umbilical cord blood (UC-MSCs), and bone marrow (BM-MSCs) were also confirmed in 3D compared to 2D culture.

[0178] Example 13: Exosome production rate according to the number of cultured cells in a 3D fibrin hydrogel culture environment

[0179] In the present invention, the production of exosomes was analyzed according to the culture density of stem cells.

[0180] Each ml of thrombin solution was mixed with 2, 10, 20, 200, 1000, 2000×10 4 After the cells were mixed separately, they were combined with 2.5 mg / ml fibrinogen solution, and then embedded in 3D fibrin hydrogel for culture according to the method proposed in Example 9, and then exosomes were isolated.

[0181] The total number of exosomes in the concentrated and purified exosomes was determined using a Nanoparticle Tracking Analyzer (model: NanoSig htPro, company brand: Malvern Panalytical, UK).

[0182] like Figure 14 As shown in Figure 2, the production rate of exosomes increases in proportion to the number of cells embedded in the hydrogel. When 10,000, 50,000, 100,000, 1 million, 5 million, and 10 million cells were embedded in each ml of hydrogel for culture, 2.6, 4.3, 8.5, 25.4, 32.9, and 12.5×10 9 exosomes.

[0183] For fibrin hydrogels, a maximum of 5 million cells per ml can be cultured. In this case, 3.3 × 10 10Exosomes. When the number of cells per ml exceeds 10 million, the hydrogel shrinks due to the cells, resulting in the hydrogel falling off the culture vessel. This causes the hydrogel to be compressed by the cells, resulting in a decrease in the production rate of exosomes.

[0184] For 2D culture environment, per cm 2 The maximum number of cells that can be cultured varies depending on the type of stem cell, but 10,000 to 50,000 cells can be cultured. However, for 3D culture environments, the maximum number of cells per cm is 2. 2 Since 10,000 to 5 million cells can be cultured, high-density culture is possible. It has been confirmed that this high-density culture method significantly increases the number of exosomes produced as the number of cells cultured per unit area increases.

[0185] Example 14: Exosome production rate according to 3D hydrogel polymer composition

[0186] In this study, we investigated the correlation between microfilament production and exosome production in stem cells within hydrogels based on the polymer type of the hydrogel. We used hydrogels composed of fibrin, collagen, gelatin, hyaluronic acid, and chondroitin as 3D cell culture matrices to investigate exosome production.

[0187] Each ml of crosslinker solution contains 2 × 10 6 The cells were suspended and mixed with 2.5 mg / ml fibrinogen, 0.2% collagen, 0.4% gelatin, 0.2% hyaluronic acid, and 0.2% chondroitin solution at a ratio of 1:1. The hydrogels were prepared by polymerization and cross-linking at 37°C for 1 hour to embed 1×10 cells per ml. 6 Fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels of myocardial-derived stem cells.

[0188] As a cell culture solution, a serum-free medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cysteine ​​in DMEM / F12 was prepared, and the prepared cell culture solution was added to the fibrin embedded with MyoCSCs and cultured for 24 hours.

[0189] Exosomes were isolated from the cell culture fluid collected 24 hours later by tangential flow filtration (TFF) using a 300 kDa molecular weight cut-off cartridge (Repligen, USA). The concentrated and purified exosomes were then analyzed using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical, UK).

[0190] like Figure 15 As shown, the microfilament lengths of cardiac stem cells embedded in fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels were 112.5, 78.5, 63.4, 58.9, and 61.8 μm, respectively, with the fibrin hydrogel exhibiting the highest microfilament length. The microfilament length of cardiac stem cells embedded in the hydrogels was proportionally correlated with the rate of exosome production.

[0191] The production of exosomes from myocardial stem cells in fibrin, collagen, gelatin, hyaluronic acid, and chondroitin hydrogels was 25.4, 12.7, 8.5, 6.4, and 7.2×10 9 In particular, the production rate of exosomes was significantly higher in myocardial-derived stem cells within fibrin and collagen hydrogels.

[0192] These results confirm that the cell stretching of stem cells embedded in hydrogels varies with the hydrogel polymer composition, reflecting that the length of microfilaments resulting from cell stretching is proportionally correlated with the rate of exosome production.

[0193] Example 15: Exosome production rate in 3D synthetic polymer and copolymer hydrogel culture environment

[0194] In the present invention, a 3D culture environment was constructed using hydrogels composed of synthetic polymer components and hydrogels copolymerized with synthetic polymers and fibrin, and myocardial stem cells were cultured. The exosome production rate was then analyzed according to the hydrogel type.

[0195] Each ml of thrombin or cross-linking agent solution contains 2 × 10 6 The cells were mixed with 2.5 mg / ml fibrinogen solution or 5% PGA, PEG, PLGA, PLLA polymer solution at a ratio of 1:1, and then the hydrogels were prepared by polymerization and cross-linking reaction at 37 °C for 1 hour to embed 1×10 cells per ml. 6 Fibrin and PGA, PEG, PLGA, and PLLA hydrogels of myocardial-derived stem cells.

[0196] In order to enhance the cell compatibility of hydrogels composed of synthetic polymers, hydrogels copolymerized with fibrin were prepared and used as 3D matrices. In order to embed myocardial stem cells in hydrogels copolymerized with fibrin and synthetic polymers, 2×10 6 Myocardial stem cells were mixed with a solution containing a crosslinker and thrombin, and then mixed with a 5% PGA, PEG, PLGA, or PLLA polymer solution containing 2.5 mg / ml fibrinogen solution at a ratio of 1:1. The mixture was then polymerized and crosslinked at 37°C for 1 hour to form 1×10 cells per ml. 6 Myocardial stem cells were embedded in hydrogels composed of fibrin copolymerized with PGA, PEG, PLGA, and PLLA.

[0197] As a cell culture solution, a serum-free medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cystein in DMEM / F12 was prepared, and the prepared cell culture solution was added to the fibrin embedded with MyoCSCs and cultured for 24 hours.

[0198] Exosomes were isolated from the cell culture fluid collected 24 hours later by tangential flow filtration (TFF) using a 300 kDa molecular weight cut-off cartridge (Repligen, USA). The concentrated and purified exosomes were then analyzed using a Nanoparticle Tracking Analyzer (NanoSight Pro, Malvern Panalytical, UK).

[0199] like Figure 16 As shown, compared with fibrin hydrogel, the degree of microfilament formation of myocardial stem cells in synthetic polymer hydrogels was 1 / 10 of that in fibrin hydrogels. The lengths of microfilaments of myocardial stem cells in hydrogels containing PGA, PEG, PLGA, and PLLA components were 13.5, 12.7, 14.6, and 13.7 μm, respectively, confirming lower microfilament formation.

[0200] However, in the case of mixed synthetic polymer and fibrin hydrogels, the lengths of myocardial stem cell microfilaments were 42.5, 58.9, 85.4, and 65.8 μm. Cell compatibility was improved by mixing with fibrin, and the degree of microfilament formation indicating cell stretching increased significantly with the increase of ligands for hydrogel matrix and cell engagement.

[0201] The production rate of myocardial stem cell exosomes in hydrogels containing synthetic polymers or a mixture of synthetic polymers and fibrin was significantly increased in hydrogels containing a mixture of synthetic polymers and fibrin. For the synthetic polymers PGA, PEG, PLGA, and PLLA, the number of exosomes produced per ml was 2.5, 1.7, 1.8, and 2.8 × 10 9 , showing a low generation rate.

[0202] For synthetic polymer hydrogels mixed with fibrin, the exosome production rate increased. When PGA, PEG, PLGA, and PLLA polymer hydrogels mixed with fibrin were cultured in a 3D matrix, the exosome production rates were 18.5, 19.7, 16.7, and 12.8 × 10 9 , showing a significant increase compared to the synthesized polymer hydrogels before mixing.

[0203] The above results confirm that cell microfilament formation is correlated with ligands that can bind to cells within the hydrogel, and that the degree of microfilament formation is closely related to the rate of exosome production from stem cells. For synthetic polymer hydrogels, reduced cell compatibility results in reduced rates of microfilament formation and exosome production.

[0204] Example 16: miRNA expression in stem cell-derived exosomes generated in 2D and 3D hydrogel culture environments

[0205] In the present invention, in order to analyze the characteristics of exosomes produced according to the culture environment, the expression level of miRNA, one of the main mediators of exosomes, was measured.

[0206] Following the method described in Example 9, myocardial stem cells were embedded in a 3D fibrin hydrogel and cultured, followed by exosome isolation. The exosomes were centrifuged at 2500 × g for 15 minutes, and miRNA was isolated from the supernatant using a miRNA isolation kit (mirvana miRNA isolation kit, Invitrogen). The exosome-extracted miRNA was used to prepare a miRNA library using reverse transcriptase (Taqman MicroRNA Reserve Transcription Kit, Invitrogen). Quantitative PCR (TaqMan MicroRNA Assay Kit) was performed using miRNA-specific target probes (Affymatrix) using the TaqMan method.

[0207] like Figure 16As shown, exosomes produced in the 3D hydrogel culture environment of the present invention showed significantly increased levels of let-7b-3b, miR126-5p, miR145-5p, miR146a-5p, and miR185-5p, miRNAs that play a role in angiogenesis, tissue regeneration, and inflammation regulation, compared to exosomes obtained in a 2D culture environment. This indicates that culturing in the 3D hydrogel of the present invention not only increases exosome production but also increases the composition of active ingredients within exosomes, thereby enhancing angiogenesis, tissue regeneration, and inflammation regulation.

[0208] Example 17: Wound repair and vascular endothelial growth factor production in 2D and 3D hydrogel culture environments Production, anti-inflammatory effects

[0209] In the present invention, in order to analyze the differences in the effects of stem cell-derived exosomes produced according to the culture environment, the effects on the growth of fibroblasts involved in wound repair, the growth of vascular endothelial cells and vascular stretching related to angiogenesis, and the effects of separating inflammatory cytokines secreted from inflammatory cells were tested.

[0210] According to the method proposed in Example 9, adipose-derived, bone marrow-derived and muscle-derived mesenchymal stem cells, peripheral nerve-derived stem cells, and myocardial-derived stem cells were respectively embedded in 3D fibrin hydrogels for culture, and then exosomes were isolated.

[0211] The effects of exosomes were evaluated on a cell-based basis using fibroblasts (3T3-L1), human umbilical vein endothelial cells (HUVECs), and RAW264.7 inflammatory cells. For the effect of exosome-induced cell growth, DFs and HUVECs cells were seeded into 96-well plates at 5,000 cells per well, and then 1.0E+08 exosomes were added to each well and cultured for 24 hours. After culture, the cells were dissolved in 0.1% SDS / TE solution and the PicoGreen dsDNA quantitation kit was used to analyze the growth rate compared to cells that were not administered with exosomes by fold ratio.

[0212] For blood vessel growth, microspheres consisting of 300 HUVEC cells were prepared, and then the microspheres were embedded in collagen gel and cultured. The length of blood vessels increased depending on whether exosomes were added or not, and the effect of exosomes on blood vessel growth was evaluated.

[0213] 100,000 RAW264.7 inflammatory cells were seeded into a 24-well plate and sensitized with 10 μg of lipopolysaccharide (LPS, Sigma). Following LPS sensitization, exosomes were added, and the levels of TNF-α and IL-β secreted by the inflammatory cells following LPS sensitization were analyzed using an ELISA kit (R&D Systems).

[0214] The results of treating the exosomes generated above in 3T3-L1 are as follows Figure 18 As shown, the stem cell-derived exosomes produced in the 3D hydrogel culture environment of the present invention significantly increased 3T3-L1 cell growth compared to a 2D culture environment. When exosomes derived from adipose, bone marrow, myocardium, peripheral nerve, and muscle stem cells were treated in the 3D culture environment, cell growth increased by 28%, 31%, 119%, 28%, and 36%, respectively, compared to exosomes produced in a 2D culture environment, demonstrating a high wound repair effect.

[0215] The results of processing exosomes produced in a three-dimensional culture environment in HUVEC cells, such as Figure 19 As shown, exosomes produced in 3D culture environments significantly induced the growth of HUVEC cells. When exosomes derived from adipose, bone marrow, myocardium, peripheral nerve, and muscle stem cells were treated in 3D culture environments, the growth of HUVECs was induced by 10%, 18%, 56%, 50%, and 38%, respectively, compared to exosomes produced in 2D culture environments.

[0216] like Figure 20 As shown, the extent of blood vessel growth in HUVEC treated with exosomes produced in a 3D culture environment showed that the lengths of HUVEC vessels derived from exosomes derived from adipose, bone marrow, myocardium, peripheral nerve, and muscle stem cells produced in 3D culture were 3.1, 2.8, 4.5, 3.5, and 4.9 mm, respectively, while those derived from exosomes produced in 2D culture were 2.7, 2.3, 3.8, 2.8, and 3.5 mm. This indicates that exosomes produced in 3D culture have significantly higher angiogenesis-inducing potential. As stem cell exosomes produced in 3D culture promote the growth of endothelial cells and blood vessels, it is expected that their angiogenic effects will be high.

[0217] like Figure 21 and Figure 22 As shown, when exosomes produced in a 3D culture environment were treated in RAW264.7 cells and then sensitized with LPS, the secretion of inflammatory cytokines TNF-α and IL-β was reduced compared to exosomes produced in a 2D culture environment.

[0218] A significant reduction in TNF-α secretion was observed in exosomes produced in 3D culture environments. When exosomes derived from bone marrow, adipose, myocardium, peripheral nerve, and muscle stem cells were treated, lower values ​​were observed compared to exosomes produced in 2D culture environments at 302, 364, 213, 306, and 254 pg / ml.

[0219] The IL-1β secretion inhibitory ability was also high when treating exosomes generated in a 3D culture environment. When treating bone marrow, adipose, myocardial, peripheral nerve, and muscle-derived stem cell exosomes, the secretion levels from RAW264.7 cells were 330, 365, 245, 305, and 287 pg / ml, respectively. However, for cells treating stem cell exosomes generated in a 2D culture environment, the secretion levels were 405, 421, 356, 364, and 358, indicating that the IL-1β secretion inhibitory effect was also high for exosomes generated in a 3D culture environment.

[0220] It was confirmed that the wound repair, angiogenesis, and anti-inflammatory effects of the above cells were significantly higher in the stem cell exosomes in the 3D culture environment, so it was confirmed that the exosome effect can be increased according to the culture environment.

[0221] Industrial Availability

[0222] Exosomes produced by the methods of the present invention have been shown to be enriched in miRNA components that regulate angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced by the methods of the present invention can be effectively used as angiogenesis, tissue regeneration, or anti-inflammatory compositions.

Claims

1. A method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching, characterized in that: include: Step (a) mixing a hydrogel in a sol state with stem cells, wherein the hydrogel in a sol state has a ligand capable of binding to the integrin of the cells and is composed of polymer chains capable of undergoing a sol-gel phase transition; as well as Step (b) is to change the hydrogel into a gel state to prepare a three-dimensional hydrogel in which stem cells are uniformly captured in three dimensions and culture the stem cells.

2. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: The method directly binds the ligands of the hydrogel to the cells without physical devices to allow the cells to stretch.

3. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: The hydrogel having a ligand capable of binding to the integrin is a natural polymer material selected from the group consisting of collagen, fibrin, gelatin, hyaluronic acid, and chondroitin, or a hydrogel copolymerized with the natural polymer material and a synthetic polymer material selected from the group consisting of polyethylene glycol, polylactic acid, polylactic-co-glycolic acid, and polyglycolic acid.

4. The method of claim 1, wherein the physical stimulation of cell stretching is used to increase the production of stem cell-derived exosomes. In the step (b), a cross-linking agent is added to the mixture of step (a) to cause the hydrogel to phase-change into a gel state.

5. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: In order to adjust the physical strength of the three-dimensional hydrogel in step (b), in step (a), any one or more polymer materials selected from the group consisting of polyethylene glycol, polylactic acid-co-glycolic acid, polyglycolic acid, chitosan, gelatin, collagen, chondroitin and hyaluronic acid are further mixed.

6. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: In order to enhance the stability by inhibiting the cellular degradation of the three-dimensional hydrogel in step (b), in step (a), a matrix metalloproteinase inhibitor capable of inhibiting any one or more matrix metalloproteinases selected from the group consisting of matrix metalloproteinase 2, i.e., MMP-2, matrix metalloproteinase 3, i.e., MMP-3, matrix metalloproteinase 8, i.e., MMP-8, and matrix metalloproteinase 9, i.e., MMP-9, and any one or more matrix metalloproteinase inhibitors selected from the group consisting of marimastat, batimastat, ilomastat, and ceprestat are further mixed.

7. The method of claim 1, wherein the physical stimulation of cell stretching is used to increase the production of stem cell-derived exosomes. In order to enhance the stability of the three-dimensional hydrogel in step (b) by inhibiting cell degradation, at least one antifibrinolytic agent selected from the group consisting of aminocaproic acid and tranexamic acid is further mixed in step (a).

8. The method of claim 1 for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching, wherein: The three-dimensional hydrogel of step (b) has a porous structure with a size of 50 nm to 300 nm, provides cell-cell bonding, and has structural properties that promote cell stretching.

9. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: The stem cells cultured in step (b) have a shape stretched in a three-dimensional axial plane, and the cells can be stretched to a length of 15 μm to 400 μm.

10. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: The method can embed 1.0×10 per ml of hydrogel at a high density. 4 ~5.0×10 6 Stem cells for culture.

11. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: In the step (b), the integrin-FAK-JNK-β-catenin signaling pathway is activated in the stem cells cultured by regulating the physicochemical properties and structural characteristics of the hydrogel.

12. The method of claim 1, wherein the physical stimulation of cell stretching is used to increase the production of stem cell-derived exosomes. In the step (b), the Wnt signaling pathway is activated in the cultured stem cells by regulating the physical properties and structural characteristics of the hydrogel, and the expression of downstream target genes of the Wnt signaling pathway is increased.

13. The method of claim 1, wherein the physical stimulation of cell stretching is used to increase the production of stem cell-derived exosomes. In the stem cell-derived exosomes prepared by the method, the composition of one or more angiogenesis-regulating miRNAs selected from the group consisting of let-7b-3b and miR126 is increased.

14. The method of claim 1, wherein the physical stimulation of cell stretching is used to increase the production of stem cell-derived exosomes. In the stem cell-derived exosomes prepared by the method, the composition of one or more inflammation-regulating miRNAs selected from the group consisting of miR145-5p, miR146a-5p, and miR185-5p is increased.

15. The method for increasing the production of stem cell-derived exosomes by transducing physical stimulation through cell stretching according to claim 1, wherein: The stem cells can be adult stem cells or mesenchymal stem cells, preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, myocardial-derived stem cells, muscle-derived stem cells, adult peripheral nerve-derived stem cells, synovial-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

16. A composition for angiogenesis, tissue regeneration, or anti-inflammation, characterized in that: The composition comprises stem cell-derived exosomes prepared by the method of any one of claims 1 to 15 as an effective 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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