Method for producing stem cell-derived exosome through cell stretching, and use thereof

Abstract

In the present invention, it has been identified that when stem cells and a sol-state hydrogel composed of a polymer that can undergo sol-gel phase transition are mixed and undergo a phase transition to a gel state, stem cells can be uniformly cultured in a 3D-state with high density. In addition, it has been identified that when stem cells are cultured by the method, the stem cells can be stretched through the regulation of the hydrogel structure so that a physical stimulus can be transmitted to the cells, thereby activating intracellular integrin-FAK and catenin-Wnt signaling and increasing exosome production efficiency and increasing functional components. Furthermore, it has been identified that the exosomes produced by the method of the present invention have increased miDNA components capable of regulating angiogenesis, tissue regeneration and inflammation. Therefore, the exosomes produced by the method of the present invention can be effectively used as a composition for angiogenesis, tissue regeneration or anti-inflammation.

Description

Method for producing stem cell-derived exosomes through cell elongation and use thereof

[0001] The present invention relates to a method for producing stem cell-derived functional exosomes by increasing cell elongation and a use thereof, and more particularly, to a method for increasing stem cell-derived exosome production by transmitting a physical stimulus through cell stretching, and a use thereof, comprising the steps of: mixing a sol-state hydrogel composed of a polymer chain capable of a sol-gel phase transition and having a ligand capable of binding to a cell's integrin; and a step of producing a 3D hydrogel in which stem cells are uniformly three-dimensionally encapsulated by subjecting the hydrogel to a gel phase transition, and culturing the stem cells.

[0002]

[0003] Stem cells possess anchorage-dependent properties, allowing them to survive and grow by attaching to culture media. Stem cell exosomes are produced by stem cells and secreted into the culture medium. These exosomes contained in the culture medium can be isolated, concentrated, and purified to produce pharmaceuticals.

[0004] To manufacture stem cell exosomes, it is essential to provide a cell attachment substrate and culture medium that enable stem cells to attach, survive, and maintain function. Conventional monolayer culture conditions (2D) require a large number of culture vessels and large volumes of culture medium for industrial-scale stem cell exosome production, resulting in low efficiency. To overcome the low efficiency of the 2D environment, which has a limited surface area for cell attachment, a technology for producing exosomes through high-density culture in a 3D environment that can provide a substrate on which many cells can attach even in a small area has been required. Matrix-dependent stem cells are prone to anoikis, a phenomenon in which cells are damaged and die when cultured in suspension without a cell attachment substrate. A 3D cell adhesion matrix is ​​required for high-density culture that can increase the volume that can be attached to cells and suppress cell death. To achieve this purpose, porous supports, hydrogels, and microspheroid carriers are being utilized as 3D cell adhesion matrices.

[0005] In the case of porous scaffolds, most cells attach and grow along the scaffold surface, and the cells grow along the scaffold surface to form a membrane, preventing the exchange of oxygen, nutrients, and waste products into the scaffold, resulting in necrosis in the center. In the case of microsphere carriers, the area where cells can attach theoretically increases compared to a 2D environment, but the disadvantages of this are that the microspheres bind to each other during suspension culture, causing aggregation, cells on the microsphere surface falling off, and long-term culture being difficult.

[0006] A phase-transition hydrogel is defined as a hydrogel that can be easily mixed with cells in a sol state before cross-linking and can undergo a phase transition to a gel state after cross-linking. The hydrogel polymer solution in a solution state before cross-linking can be uniformly mixed with cells, and cells can be uniformly distributed in 3D within the hydrogel after cross-linking. In addition, compared to porous supports or microsphere carriers, it has the advantage of good interpenetrating network (IPN) between polymer chains, which allows for smooth movement of oxygen, nutrients, and metabolites.

[0007] However, hydrogels have fragile biochemical properties that are degraded by matrix metalloproteinases (MMPs) secreted by cells, and if the physical properties are too hard, cell binding and elongation are inhibited, resulting in a decline in the molecular biological functions of cells. The interaction between cells and polymer chains in hydrogels is greatly affected by the components of the constituent polymers. In addition, since the structural characteristics and physical properties of the hydrogel change depending on the concentration, molecular weight, and degree of crosslinking of the polymers constituting the hydrogel, cell growth, migration, activity, elongation, and gene expression also change depending on the structure and physical properties of the hydrogel. As a result, the biological functions and activities of the cells are changed, and the rate of exosome production and the composition of genes in the exosomes are also greatly affected.

[0008] There is a need for a technology that can control the structure and physical properties of 3D hydrogels to act as a cell adhesion matrix that enables high-density cell culture in a 3D culture environment, and to optimize the interaction between polymer chains within the hydrogel and cells, thereby enhancing the production of exosomes from stem cells and the production of exosomes with enhanced biological functions.

[0009]

[0010] Accordingly, in the present invention, as a result of extensive efforts to efficiently produce stem cell-derived exosomes by controlling the structure and physical properties of the hydrogel, it was confirmed that stem cells can be uniformly and densely cultured in 3D within the 3D hydrogel by mixing stem cells with a sol-state polymer solution capable of a sol-gel phase transition, which is composed of a polymer chain having a ligand capable of binding to the integrin of a cell, and performing a cross-linking reaction followed by a gel phase transition. By providing a 3D cell adhesion matrix to stem cells using a hydrogel, an environment can be provided in which cells can be cultured at a density of 10 to 100 times greater per area than a 2D culture environment, thereby enabling the culture of a large number of cells even with a small number of culture vessels, thereby improving the efficiency of exosome production.

[0011] Furthermore, culturing stem cells within 3D hydrogels provides a method for controlling gene expression and function of cells by controlling the interaction between stem cells and the hydrogel through modulating the physicochemical properties and structural characteristics of the hydrogel, including pore size. Depending on the hydrogel properties, the physiological activity, function, gene expression, exosome production rate, and exosome composition of the cells are altered.

[0012] The present invention provides a method for stretching cells by controlling the physicochemical and structural properties of a hydrogel, inducing activation of the integrin-FAK signaling pathway depending on the degree of cell stretching, and thereby increasing the exosome production rate of the cells. In addition, the present invention provides a method for maximizing cell-cell bonding by controlling the hydrogel structure, activating the β-catenin-Wnt signaling pathway to activate intracellular gene expression, and thereby controlling the miRNA composition within exosomes produced from the cells.

[0013] The present invention provides a culture base for manufacturing exosomes at an industrial level through high-density culture of stem cells in a 3D hydrogel, and provides a method for manufacturing a composition that can increase the exosome production rate through cell elongation and enhance anti-inflammation, tissue regeneration, and angiogenesis in exosomes.

[0014]

[0015] To achieve the above-mentioned purpose,

[0016] The present invention comprises the steps of (a) mixing a hydrogel in a sol state, which has a ligand capable of binding to an integrin of a cell and is composed of a polymer chain capable of a sol-gel phase transition, and stem cells; and

[0017] (b) A method for increasing the production of stem cell-derived exosomes by delivering a physical stimulus through cell stretching, including a step of manufacturing a 3D hydrogel in which stem cells are uniformly captured in a three-dimensional manner by phase-transforming the hydrogel into a gel state and culturing stem cells, is provided.

[0018]

[0019] In a preferred embodiment of the present invention, the method can stretch cells by directly binding the ligand of the hydrogel to the cells without a physical device.

[0020] In another preferred embodiment of the present invention, the hydrogel having a ligand capable of binding to the integrin may be a hydrogel copolymerized with a natural polymer selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin; or a synthetic polymer selected from the group consisting of PEG (Polyethylene Glycol), PLA (Polylactic Acid), and PGA (Polyglycolic Acid).

[0021] In another preferred embodiment of the present invention, step (b) may add a cross-linking agent to the mixture of step (a) to cause a phase transition of the hydrogel into a gel state.

[0022] In another preferred embodiment of the present invention, in order to strengthen the physicochemical properties of the 3D hydrogel of step (b), at least one polymer material selected from the group consisting of PEG, PLGA, PGA, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid may be additionally mixed in step (a), and through the strengthening of the properties, "gel compaction" caused by the hydrogel being detached from the culture vessel due to the contractile force of cells can be prevented.

[0023] In another preferred embodiment of the present invention, in order to enhance stability by inhibiting degradation by cells of the 3D hydrogel in step (b), at least one matrix metalloproteinase inhibitor (MMP) selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9, and at least one MMP inhibitor selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat, which can inhibit MMP, may be additionally mixed in step (a).

[0024] In another preferred embodiment of the present invention, in order to enhance stability by suppressing degradation by cells of the 3D hydrogel of step (b), one or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid may be additionally mixed in step (a).

[0025] 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, and thus can have structural properties that provide cell-cell bonding and promote cell stretching.

[0026] In another preferred embodiment of the present invention, the stem cells cultured in step (b) have an appearance of being extended in a 3D axial plane, and the cells can be extended to a length of 15 µm to 400 µm.

[0027] In another preferred embodiment of the present invention, the method comprises the steps of: 4 ~ 5.0ⅹ10 6Stem cells can be cultured by incorporating them at high density.

[0028] In another preferred embodiment of the present invention, the stem cells cultured in step (b) can have the integrin-FAK-JNK-β-catenin signaling pathway activated through the control of the hydrogel physicochemical properties and structural characteristics.

[0029] In another preferred embodiment of the present invention, by controlling the hydrogel properties and structural characteristics in step (b), the cultured stem cells can activate the Wnt signaling pathway and increase the expression of Wnt signaling downstream target genes.

[0030] In another preferred embodiment of the present invention, the stem cell-derived exosome prepared by the above method may have an increased composition of at least one angiogenesis-regulating miRNA selected from the group consisting of let-7b-3b and miR126; or at least one inflammation-regulating miRNA selected from the group consisting of miR145-5p, miR146a-5p, and miR185-5p.

[0031] In another preferred embodiment of the present invention, the stem cells may be adult stem cells or mesenchymal stem cells, and preferably may be adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, muscle-derived stem cells, myocardium-derived stem cells, peripheral nerve-derived stem cells, synovial membrane-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

[0032]

[0033] To achieve other purposes,

[0034] The present invention provides a composition for angiogenesis, tissue regeneration, or anti-inflammation comprising stem cell-derived exosomes prepared by the above method as an active ingredient.

[0035] In a preferred embodiment of the present invention, the composition may be a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

[0036]

[0037] The present invention confirmed that by mixing a sol-state hydrogel composed of a polymer capable of sol-gel phase transition and stem cells and performing phase transition to a gel state, stem cells can be uniformly cultured at a high density in a 3D state. In addition, when stem cells are cultured by the above method, the stem cells can be stretched through adjustment of the hydrogel structure to transmit physical stimuli to the cells, thereby activating intracellular integrin-FAK and catenin-Wnt signaling, thereby increasing the efficiency of exosome production and functional components. It was confirmed that exosomes produced by the method of the present invention increase miRNA components that can regulate angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced by the method of the present invention can be usefully utilized as compositions for angiogenesis, tissue regeneration, or anti-inflammation.

[0038]

[0039] Figure 1 is a photograph of the cell characteristics observed in a 2D culture environment of substrate-dependent stem cells. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; CB-MSC, 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, synovial membrane-derived mesenchymal stem cells; UC-MSC, umbilical cord-derived mesenchymal stem cells.

[0040] Figure 2 is a flow cytometry histogram photograph observing the immunophenotypic characteristics of myocardial stem cells.

[0041] Figure 3 is an image observing the differentiation characteristics of matrix-dependent stem cells into adipocytes and osteoblasts. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, myocardium-derived stem cells; PNSC, peripheral nerve-derived stem cells.

[0042] Figure 4 shows photographs and results for (A) hydrogel porous structure and (B) pore size according to fibrinogen concentration.

[0043] Figure 5 shows data measuring the degree of microfilament formation, which indicates the degree of cell elongation (A) in a fibrin hydrogel according to fibrinogen concentration, and (B) the microfilament area per cell.

[0044] Figure 6 is a photograph of the expression of integrin-β1 and p-FAK of cardiac stem cells in fibrin hydrogel according to the fibrinogen concentration.

[0045] Figure 7 shows (A) Western blot images of the expression intensities of p-FAK, p-JNK, and p-β-catenin in cardiac stem cells in 3D fibrin hydrogels according to fibrinogen concentration, and (B) the measured values ​​of the expression rates of p-FAK, p-JNK, and p-β-catenin corrected to the expression intensities of cardiac stem cells in a 2D culture environment. *, p < 0.05 compared to the expression rate in a 2D culture environment; **, p < 0.01 compared to the expression rate in a 2D culture environment.

[0046] Figure 8 shows the results of the expression of Wnt signaling mRNAs in cardiac stem cells in 3D fibrin hydrogels, corrected to the mRNA expression rate of cardiac stem cells in a 2D culture environment. (A) Expression rates of Wnt ligand-related mRNAs, (B) Wnt receptor-related mRNAs, and (C) downstream mRNAs targeted by the Wnt signaling pathway. *, p < 0.05 compared to 2D culture; **, p < 0.01 compared to 2D culture.

[0047] Figure 9 is a graph showing (A) the expression of exosome markers CD9, CD63, and CD81 (red) and pFAK expression (green) observed by immunofluorescence staining to measure the exosome production rate of cardiac stem cells in 3D fibrin hydrogels according to fibrinogen concentration, and (B) the quantification of immunofluorescence intensity. **, p < 0.01 compared to fibrin hydrogels prepared with 5 mg and 10 mg fibrinogen.

[0048] Figure 10 shows data for measuring the exosome production rate of cardiac stem cells in 3D fibrin hydrogel and 2D culture environments according to fibrinogen concentration, (A) the number of exosomes per ml measured using a nanoparticle tracking analyzer, and (B) the protein content. *, p < 0.01 compared to 2D culture environment.

[0049] Figure 11 shows the diameter of exosomes from cardiac stem cells in 3D fibrin hydrogel and 2D culture environments according to fibrinogen concentration, measured using a nanoparticle tracking analyzer, and the measured values ​​for (A) average diameter and (B) mode diameter of exosomes.

[0050] Figure 12 is a flow cytometry dot plot image for analyzing the immunophenotypic characteristics of myocardial stem cell exosomes generated in a 3D fibrin hydrogel and a 2D culture environment.

[0051] Figure 13 shows the measured values ​​analyzing the exosome production rate according to stem cell sources in 3D fibrin hydrogel and 2D culture environments. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; CB-MSC, 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, synovial membrane-derived mesenchymal stem cells; UC-MSC, umbilical cord-derived mesenchymal stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0052] Figure 14 shows the measured values ​​for the exosome production rate according to the seeding density of myocardial stem cells in 3D fibrin hydrogels. *, p < 0.05 vs. 1.0E+04; **, p < 0.01 vs. 1.0E+04.

[0053] Figure 15 shows the results for (A) microfiber formation and (B) exosome production rate of cardiac stem cells in a 3D culture environment according to the natural polymer components of the hydrogel composition. *, p < 0.05 compared to gelatin, hyaluronan, and chondroitin; **, p < 0.01 compared to gelatin, hyaluronan, and chondroitin.

[0054] Figure 16 shows the measured values ​​for (A) microfiber formation and (B) exosome production rates of cardiac 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.

[0055] Figure 17 shows the miRNA expression characteristics in exosomes of myocardial stem cells generated in 3D fibrin hydrogels and 2D culture environments. The miRNA expression rate is expressed as a ratio corrected to that of bone marrow-derived mesenchymal stem cells (BM-MSCs). *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0056] Figure 18 is a graph comparing the fibroblast growth-inducing ability of stem cell exosomes generated in 3D fibrin hydrogels and 2D culture environments. The comparison was made by adjusting the cell counts of the group in which the vehicle-free culture medium was added. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiac stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0057] Figure 19 is a graph comparing the ability of stem cell exosomes generated in 3D fibrin hydrogels and 2D culture environments to induce vascular endothelial cell growth. The comparison was made by adjusting the cell counts of the group in which the vehicle-free culture medium was added. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiac stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0058] Figure 20 shows the results of measuring the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to induce vascular endothelial cell growth. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, myocardium-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0059] Figure 21 shows the results of measuring the inhibitory ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to suppress inflammatory cell TNF-α secretion. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, myocardium-derived stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0060] Figure 22 shows the results of measuring the ability of stem cell exosomes produced in 3D fibrin hydrogels and 2D culture environments to suppress inflammatory cell IL-1β secretion. AT-MSC, adipose-derived mesenchymal stem cells; BM-MSC, bone marrow-derived mesenchymal stem cells; MyoCSC, cardiac stem cells; PNSC, peripheral nerve-derived stem cells; SM-MSC, muscle-derived stem cells; *, p < 0.05 compared to 2D culture environment; **, p < 0.01 compared to 2D culture environment.

[0061]

[0062] Hereinafter, the present invention will be described in detail.

[0063] The present invention, in a consistent viewpoint, comprises the steps of (a) mixing a hydrogel in a sol state, which has a ligand capable of binding to an integrin of a cell and is composed of a polymer chain capable of a sol-gel phase transition, and stem cells; and

[0064] (b) A method for increasing the production of stem cell-derived exosomes by delivering a physical stimulus through cell stretching, including a step of manufacturing a 3D hydrogel in which stem cells are uniformly captured in a three-dimensional manner by phase-transforming the hydrogel into a gel state and culturing stem cells.

[0065] More specifically, the present invention comprises the steps of (a) mixing a hydrogel in a sol state, which has a ligand capable of binding to an integrin of a cell and is composed of a polymer chain capable of a sol-gel phase transition, and stem cells;

[0066] (b) a step of manufacturing a 3D hydrogel in which stem cells are uniformly captured in three dimensions by changing the hydrogel to a gel state and culturing the stem cells;

[0067] (c) a step of increasing the rate of stem cell-derived exosome production by stretching the cell through 3D bonding between cell-polymer chains to transmit physical stimulation into the cell and strengthening the integrin-FAK signaling pathway; and

[0068] (d) It relates to a method for increasing miRNA components that act on anti-inflammation, tissue regeneration, and angiogenesis in stem cell-derived exosomes by strengthening the β-catenin-Wnt signaling pathway through cell-cell interaction.

[0069]

[0070] In the present invention, the stem cell may be an adult stem cell or a mesenchymal stem cell, and preferably may be an adipose-derived mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, an umbilical cord blood-derived mesenchymal stem cell, a myocardium-derived stem cell, a muscle-derived stem cell, a peripheral nerve-derived adult stem cell, a synovial membrane-derived mesenchymal stem cell, or an umbilical cord-derived mesenchymal stem cell.

[0071] In the present invention, the method may be characterized in that the ligand of the hydrogel binds to the cells without a physical device, thereby stretching the cells.

[0072] In the present invention, the hydrogel having a ligand capable of binding to the integrin is a natural polymer selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin; or

[0073] It may be characterized by being a hydrogel copolymerized with the above natural polymer material and a synthetic polymer material selected from the group consisting of PEG (Polyethylene Glycol), PLA (Polylactic Acid), and PGA (Polyglycolic Acid), and preferably fibrinogen, collagen, or gelatin.

[0074] In the present invention, the step (b) may be characterized by adding a cross-linking agent to the mixture of step (a) to cause a phase transition of the hydrogel into a gel state.

[0075]

[0076] The above polymers have a native ligand that can directly bind to cell integrin, and thus can be applied to the present invention.

[0077] Among these polymers, collagen, fibrin, and gelatin can be controlled in terms of polymer content and cross-linking density, allowing for the control of the microstructure and physical properties within the hydrogel, and as a result, the production rate and composition of cell exosomes can be controlled.

[0078] On the other hand, the hydrogel has a biodegradable property that is easily degraded by matrix metalloproteinases (MMPs) secreted by cells. In addition, the weak physical properties that cause the hydrogel to shrink due to cell-mediated contraction after the polymer chains bind to the cells, thereby losing its structural function as a 3D matrix, have emerged as a drawback. To overcome these biochemical and physical shortcomings, technologies to enhance biodegradation and structural properties are required, and stability against biodegradation can be increased by introducing MMP inhibitors or antifibrinolytic agents. In addition, resistance to cell-mediated contraction can be increased by using hydrogels with enhanced physical properties through copolymerization with polymers such as PEG, PLGA, PGF, chitosan, and hyaluronic acid, which are not degraded by MMPs and have high physical strength.

[0079]

[0080] In order to control the physical strength of the 3D hydrogel of step (b), at least one polymer material selected from the group consisting of PEG, PLGA, PGF, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid may be additionally mixed into step (a).

[0081] In addition, in order to enhance stability by inhibiting degradation by cells of the 3D hydrogel in step (b), at least one matrix metalloproteinase inhibitor selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat, which can inhibit at least one matrix metalloproteinase selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9 in step (a); or

[0082] One or more antifibrinolytics selected from the group consisting of aminocaproic acid and tranexamic acid may be additionally mixed.

[0083]

[0084] In the present invention, the 3D hydrogel of step (b) may be characterized by having a porous structure with a size of 50 nm to 300 nm, and thus has structural characteristics capable of supporting cell-cell bonding and simultaneously promoting cell stretching.

[0085]

[0086] The stem cells cultured in the above step (b) have an appearance of extending in a 3D axial plane, and the cells can be characterized by being able to extend to a length of 15 ㎛ to 400 ㎛. In addition, the method can be characterized by 1.0×10 per ㎖ of hydrogel. 4 ~ 5.0ⅹ10 6 Stem cells can be cultured by incorporating them at high density.

[0087] Since hydrogels can provide a 3D cell attachment matrix to which cells can attach at a high density, the present invention can provide a method for increasing the efficiency of exosome production by culturing at a higher density than conventional 2-dimensional (2D) culture through high-density culture. In the case of lipid-dependent cells such as mesenchymal stem cells, the density can be increased to 5.0×10 per cm2 in a 2D environment. 4 While cell density cannot be higher than 0.5 to 5.0 × 10 per cm3 in 3D environment, 6 By being able to culture the above cells, it is possible to provide a cell attachment substrate that can culture cells at a high density 10 to 100 times higher than 2D.

[0088] The rate of exosome production is directly proportional to the number of cells producing exosomes. In particular, in the case of stem cells, which are anchorage-dependent cells, the area of ​​the substrate to which cells can be attached is an absolute variable that determines the number of cells that can be cultured, and the yield of exosome production is determined according to the number of cultured cells. The present invention provides a technology that can produce exosomes on a large scale even in a small-sized culture vessel by providing a 3D substrate on which cells can be attached at a high density using a hydrogel. In particular, there is an advantage in that stem cells can be uniformly incorporated into the 3D hydrogel by mixing the cells in a sol state and converting the phase to a gel state using a sol-gel phase transition hydrogel. When stem cells are cultured in a T175 flask, the area of ​​the flask is 175 cm. 2 The maximum number of stem cells that can be cultured is 7 to 8E+06, but the number of cells that can be cultured in a 1 cm thick 3D hydrogel is up to 8E+08.

[0089]

[0090] Specifically, the present invention can control the exosome production rate of stem cells and the function of inducing inflammation, tissue regeneration, and angiogenesis by controlling the structure and physicochemical properties of the hydrogel. It provides a method for inducing cell stretching by combining polymer chains forming the nanostructure in the hydrogel with cells, and as a result, activating integrin, FAK, β-catenin, and Wnt signaling by physical stimulation compared to a 2D environment. It provides a method for controlling cell function by controlling the structure and physicochemical properties of the hydrogel, and ultimately increasing the efficiency of stem cell exosome production and strengthening functional components. The loose nanostructure in the hydrogel increases the cell stretching and cell-to-cell contact of stem cells, and the stem cells in the dense nanostructure decrease the cell stretching and cell-to-cell contact. Depending on the degree of cell elongation and cell-cell adhesion, the activity of FAK, integrin, β-catenin, and Wnt signaling pathways changes, which in turn affects the rate of exosome production in stem cells and the composition of functional components in exosomes.

[0091] In a specific embodiment of the present invention, a hydrogel is prepared using fibrinogen, and specifically, stem cell-derived exosomes can be prepared by a method including the steps of (1) mixing a hydrogel polymer solution in a sol state with stem cells; and (2) adding a cross-linking agent to the mixture to prepare a gel-state hydrogel in which stem cells are uniformly encapsulated in a 3D manner, and culturing the stem cells.

[0092] Furthermore, hydrogels can have their structural characteristics and physicochemical properties controlled by the polymer content and degree of cross-linking. Higher polymer content results in a more rigid structure, while lower content results in a looser structure. However, the shortcomings of loose hydrogels can be addressed by adding MMP inhibitors, antifibrinolytic agents, and synthetic polymers, which can enhance the stability of loose hydrogels against degradation and compensate for their weak physical strength, which can cause hydrogels to compact and detach from the culture vessel due to cell shrinkage.

[0093]

[0094] In another specific embodiment of the present invention, it was confirmed that the structural characteristics and physical properties of the fibrin hydrogel can be controlled by controlling the fibrinogen concentration, and that as the fibrinogen concentration increases, the pore size decreases, so that the pore spacing becomes narrower and a dense and solid structure can be manufactured, and that as the fibrinogen content decreases, a hydrogel with a loose structure can be manufactured.

[0095] That is, in the present invention, when a hydrogel was prepared with fibrinogen at a concentration of 1.25 mg / ml to 5 mg / ml, the 3D hydrogel had a porous structure with a size of 50 nm to 300 nm, preferably 100 nm to 300 nm, and it was confirmed that stem cells in the fibrin hydrogel composed of a low concentration of fibrinogen were elongated to a length of 15 μm to 400 μm and that cell-cell bonding was increased (Figs. 4 and 5).

[0096] In addition, when stem cells were cultured using the fibrin hydrogel manufactured in the present invention, it was confirmed that the FAK, integrin, Wnt, and β-catenin cell signaling pathways were activated in proportion to the elongation of stem cells and the degree of cell-cell bonding in the fibrin hydrogel with a loose structure (Figs. 6 to 9), and that exosome production increased (Figs. 10 and 11).

[0097]

[0098] Furthermore, when comparing exosomes produced in the 3D hydrogel culture environment of the present invention with exosomes produced in 2D culture, there was no significant difference in the size and immunophenotypic characteristics of the exosomes (Fig. 12), but it was confirmed that the production of exosomes depending on the stem cell source (Fig. 13) and the expression levels of miRNAs regulating angiogenesis, tissue regeneration, and anti-inflammation significantly increased (Fig. 17). In addition, it was confirmed that the angiogenesis, tissue regeneration, and inflammation control effects of exosomes produced in the 3D hydrogel culture environment of the present invention were significantly increased compared to exosomes produced in 2D culture (Figs. 18 to 22).

[0099]

[0100] In the present invention, the degree of stem cell elongation can be controlled by adjusting the nanostructure of the hydrogel, and it was confirmed that as cell elongation increases, physical stimulation activates the cell signaling pathway of the stem cells, thereby increasing exosome production. In addition, it was confirmed that the exosomes produced by the method of the present invention increased the types of proteins and miRNAs that regulate cell growth, wound healing, angiogenesis, and inflammation within the exosomes, thereby enhancing the angiogenesis, tissue regeneration, and anti-inflammatory effects of the exosomes.

[0101]

[0102] From another consistent perspective, the present invention relates to a composition for angiogenesis, tissue regeneration, or anti-inflammatory purposes, which comprises stem cell-derived exosomes prepared by the above method as an effective ingredient.

[0103] In the present invention, the composition may be a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

[0104]

[0105] The above angiogenic composition can induce or increase the migration of vascular endothelial cells and promote angiogenesis in diseases requiring angiogenesis.

[0106] The disease requiring the above angiogenesis may be at least one selected from the group consisting of wounds, burns, ulcers, necrosis, arteriosclerosis, angina pectoris, myocardial infarction, cerebrovascular disease, and alopecia.

[0107]

[0108] The above tissue regeneration composition can be used to regenerate tissues selected from, but not limited to, skin, cartilage, bone, blood vessels, brain, liver, heart, ligaments, muscles, spinal cord, blood, bone marrow, lungs, teeth, nerves, cornea, retina, esophagus, spine, kidney, pancreas, or urethra. Furthermore, the tissue regeneration composition can be used to improve skin condition, fill wrinkles, or shape facial or body contours. Furthermore, it can be usefully used as a dermal filler.

[0109]

[0110] The above anti-inflammatory composition may be a pharmaceutical composition for preventing or treating an inflammatory disease, and the inflammatory disease may 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.

[0111] In the present invention, the inflammatory disease may 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, systemic lupus erythematosus (SLE), atherosclerosis, asthma, arteriosclerosis, edema, rheumatoid arthritis, delayed-onset allergy (type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, arthritis, cystic fibrosis, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy.

[0112]

[0113] The pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the forms of topical preparations, suppositories, and sterile injectable solutions. Depending on each dosage form, pharmaceutically acceptable carriers, excipients, and diluents may be further included. In addition, it can be formulated and used in the forms of topical preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and sterile injectable solutions according to conventional methods.

[0114] The carrier, excipient and diluent include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, etc. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants that are commonly used.

[0115] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0116] The term "administration" as used herein means providing the pharmaceutical composition of the present invention to a subject by any suitable method. The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount, which is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue, animal, or human as contemplated by a researcher, veterinarian, physician, or other clinician, i.e., an amount that induces alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by those skilled in the art and can be adjusted according to various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the composition, the treatment period, and concurrently used drugs. The pharmaceutical composition of the present invention may be administered to a subject by various routes. For example, it can be administered intravenously, intraperitoneally, intramuscularly, intraarterially, orally, intracardiacly, 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 be administered once a day or divided into several doses.

[0117]

[0118] The term "quasi-drug" as used herein refers to products used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing human or animal diseases, and those with milder effects than pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, "quasi-drug" refers to products other than those used for pharmaceutical purposes, and includes products used to treat or prevent human or animal diseases, as well as products with mild or no direct effects on the human body.

[0119] The pharmaceutical composition of the present invention is not particularly limited in its formulation, and may be, for example, a cosmetic composition having the formulation of a softening toner, a nourishing toner, a massage cream, a nourishing cream, a pack, a mask pack, a mask sheet, a gel, or a skin adhesive type cosmetic, and may also be a transdermal administration formulation such as a lotion, ointment, gel, cream, patch, or spray.

[0120] Additionally, for each formulation, the quasi-drug composition may optionally select and combine other ingredients according to the formulation or intended use of other quasi-drugs. The amount of active ingredients mixed may be appropriately determined depending on the intended use (inhibition or relief). For example, the composition may include conventional excipients such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0121]

[0122] 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 cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray, but is not limited thereto.

[0123] In addition, the cosmetic composition of the present invention may further contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. In addition, the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0124]

[0125] These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0126]

[0127] Example 1: Stem cell isolation and culture

[0128] In the present invention, in order to produce stem cell-derived exosomes, various stem cells were isolated and cultured.

[0129] Adipose-derived mesenchymal stem cells (AT-MSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), skeletal muscle-derived mesenchymal stem cells (SM-MSCs), cardiac muscle-derived mesenchymal stem cells (MyoCSCs), peripheral nerve-derived stem cells (PNSCs), synovial membrane-derived mesenchymal stem cells (Syn-MSCs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs) were isolated and cultured using a three-dimensional organ culture method.

[0130] 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. The adipose tissue (AT), bone marrow (BM), myocardium (MY), skeletal muscle (SM), synovium (SY), peripheral nerve (PERI), and umbilical cord (UC) tissues to be incorporated into the hydrogel were cut into 0.2 to 2 mm pieces using a scalpel. 3After cutting into small pieces, the tissue hematoma and fibrous tissue were removed before taking the pieces. The tissue pieces were washed three times with PBS to remove blood cells in the tissue, washed, and then mixed with 1 ml of thrombin solution per 10 mg of tissue piece and placed in an ice bucket for 10 minutes. After that, an equal amount of fibrinogen solution was added and mixed thoroughly, and the mixture was transferred to a culture dish and placed in a 37°C incubator for 1 hour to form a gel. The pieces were embedded in a fibrin hydrogel with a final wound repair matrix composition of 0.25% fibrinogen and 0.5 unit / ml thrombin by adding fibrinogen solution.

[0131] The culture medium for organ culture was composed of 45% (vol / vol) DMEM, 45% Ham's F12, 10% fetal bovine serum (FBS; Invitrogen), 20 ng / mL EGF, 2 ng / mL bFGF, 10 ng / mL IGF, 10 μg / mL gentamicin (Invitrogen), and 200 mg / mL tranexamic acid. Twice the volume of the gel was added to the organ culture medium, and after adding the organ culture medium, the culture vessel was placed on an orbital shaker and slowly shaken at 30 rpm for 14 days. The organ culture medium was replaced with fresh organ culture medium twice a week.

[0132] After two weeks of long-term culture, the culture medium was removed, DMEM was added, and the tissue sections and the remaining long-term culture medium in the wound repair matrix were removed by washing three times for 10 minutes.

[0133] Fresh organ culture medium without PAI was added, and the culture vessel was placed on an orbital shaker and stirred at 37°C and 30 rpm for 1 hour to release the stem cells that had migrated and grown in the wound repair matrix into the culture medium. The stem cells released into the culture medium were collected and transferred to a 50 ml centrifuge tube and centrifuged at 300 x g for 10 minutes. After removing the supernatant and dispersing it in 10 ml cell culture medium, the cell number was calculated using a hemocytometer, and cm 2 Stem cells were expanded and cultured in a monolayer culture environment by seeding them in culture vessels at a density of 3,000 cells per cell.

[0134] Umbilical cord blood was purified by Ficoll gradient centrifugation to isolate mononuclear cells. The cells were washed three times by adding PBS and centrifuging. The cells were then suspended in cell culture medium, seeded, and cultured for two weeks. Colonies formed after culture were trypsinized, dissociated, and expanded using conventional monolayer culture methods. All stem cells used for exosome production were harvested after passage 4.

[0135]

[0136] As shown in [Figure 1], all cells isolated and cultured from fat, bone marrow, umbilical cord blood, myocardium, skeletal muscle, peripheral nerve, synovium, and umbilical cord show substrate-dependent characteristics of attachment and growth on an attachment vessel, and typical characteristics of stromal cells with a spindle-shaped shape can be confirmed.

[0137]

[0138] Example 2: Analysis of immunophenotypic characteristics of stem cells

[0139] After long-term culture, the immunophenotypic characteristics of stem cells expanded using conventional monolayer culture were analyzed. Ten thousand cells were reacted with anti-human antibodies conjugated to fluorescent markers for CD31, CD34, CD44, CD45, CD73, and CD105. The positivity for each antibody was analyzed using a FACSCalibur (Becton Dickinson, USA) flow cytometer (hereafter referred to as FCM), and the expression rate was analyzed in more than ten thousand cells.

[0140]

[0141] Stem cell marker expression rate (%)Primary AbAT-MSCsBM-MSCsCB-MSCsMyoCSCsPNSCsSM-MSCsSyn-MSCsUC-MSCsCD31(%)0.81.10.90.21.40.51.10.8CD34(%)1.21.31.10.40.80.81.10.8CD44(%)96.493.4 92.594.692.696.895.792.4CD45(%)0.91.50.51.21.41.11.41.1CD73(%)95.19 3.891.496.097.593.897.596.5CD105(%)93.496.193.494.896.893.198.197.5

[0142] As shown in [Figure 2] and [Table 1], stromal-dependent cells isolated and cultured from adult tissues expressed stromal cell markers CD44, CD73, and CD105 positively in over 90% of cells. However, vascular endothelial cell, hematopoietic cell, and immune cell markers CD31, CD34, and CD45 were expressed at less than 2%, indicating that the cells used for exosome production had a purity of over 90% stromal cells.

[0143]

[0144] Example 3: Confirmation of differentiation characteristics of stem cells into adipocytes and osteoblasts.

[0145] To evaluate the ability of stem cells to differentiate into adipocytes, 200,000 cells were seeded in 24-multiwell tissue culture plates and cultured for 14 days in a medium containing 90% DMEM, 10% CS, 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma), 80 μM indomethacin, 1 μM dexamethasone, and 5 μg / ml insulin. Differentiation of cells into adipocytes was determined by staining with 0.5% Sudan Black (Sigma), an indicator of intracellular lipid accumulation, for 1 hour at room temperature to determine whether lipids accumulated in the cytoplasm.

[0146]

[0147] To evaluate the osteoblast differentiation capacity of stem cells, 200,000 cells were seeded in 24-multiwell tissue culture plates and cultured for 14 days in a medium containing 90% α-MEM, 10% CS, 50 μg / mL ascorbic acid, 0.1 μM dexamethasone (Sigma), and 1 mM calcium glycerophosphate (Sigma). Osteoblast differentiation of stem cells was evaluated by staining mineral accumulation with 1 μM calcein and then using a fluorescent microscope.

[0148]

[0149] As shown in [Figure 3], differentiation into adipocytes and osteoblasts was demonstrated in matrix-dependent stem cells. Differentiation into adipocytes was confirmed by accumulation of intracytoplasmic lipid vesicles and positive staining with Sudan Black.

[0150] Differentiation into osteoblasts was observed by phase contrast microscopy with mineral crystals in the generated extracellular matrix, and it was confirmed by calcein fluorescence microscopy that these minerals were calcium phosphate.

[0151] Although the degree of differentiation into adipocytes or osteoblasts varied depending on the tissue of origin, differentiation into adipocytes and osteoblasts was confirmed in all stem cells, confirming that they are stem cells with multipotent differentiation capacity.

[0152]

[0153] Example 4: Control of hydrogel structure according to polymer concentration

[0154] Fibrinogen (Sigma) was dissolved in DMEM and fibrinogen solutions were prepared at four concentrations: 2.5, 5.0, 10.0, and 20.0 mg / mL. Aminomethylbenzoic acid (Sigma) was added at a concentration of 100 μg / mL to the fibrinogen solutions. Thrombin (Sigma) was dissolved in DMEM and a 1 unit / mL thrombin solution was prepared. To confirm the size and connectivity of the fibrin pores, a fibrinogen solution was prepared by mixing Alexa Fluor 546-conugated fibrinogen (Molecular Probes) in a 1:10 ratio, and then mixed with a 1 unit / mL thrombin solution in a 1:1 ratio. Fibrin hydrogels were prepared through polymerization and cross-linking reactions at 37°C for 1 hour. The structural properties of fibrin hydrogels produced at four fibrinogen concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) were evaluated using a confocal microscope (Zeiss LSM 900).

[0155]

[0156] As shown in [Figure 4], as the concentration of fibrinogen, a constituent polymer, increased, the pore size of the manufactured fibrin hydrogel decreased and a hydrogel with a dense porous structure was formed.

[0157] On the other hand, as the fibrinogen concentration decreased, the pore size decreased and a hydrogel with a coarse structure was formed. As shown in [Figure 4B], the pore sizes of the fibrin hydrogels prepared with fibrinogen solutions at four concentrations (1.25, 2.5, 5.0, and 10.0 mg / mL) were 265.7, 165.2, 76.8, and 38.5 nm, respectively. As the fibrinogen concentration increased, the pore diameter significantly decreased and the hydrogels exhibited faithful structural characteristics.

[0158]

[0159] Example 5: Cell elongation according to hydrogel nanostructure

[0160] The interaction between hydrogel physical and structural properties and stem cells was evaluated by 3D culture of myocardial stem cells (MyoCSCs).

[0161] One ml of fibrinogen solutions at four concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) were mixed with one million MyoCSCs, and then each was mixed with the same volume of thrombin solution. Fibrin hydrogels containing MyoCSCs were prepared through polymerization and cross-linking reactions at 37°C for 1 hour. A serum-free medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cysteine ​​in DMEM / F12 was prepared as a cell culture solution, and the prepared cell culture solution was added to the fibrin containing MyoCSCs and cultured for 12 hours.

[0162] After culture, the stem cell-incorporated hydrogel was fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, the cells were reacted with Alexa Fluor 488-conjugated phalloidin (Invitrogen) for 60 minutes. The length of microfilaments formed within the cytoplasm was measured using a confocal microscope to evaluate the degree of cell elongation.

[0163]

[0164] As shown in [Figure 5], the degree of cell elongation varied depending on the type of fibrin hydrogel and the presence of intracellular microfibers. Unlike their appearance in a 2D culture environment, stem cells within hydrogels exhibited 3D axial cell elongation, and their cytoplasm exhibited a characteristically long and thin shape.

[0165] The degree of cell elongation of stem cells in loose fibrin hydrogels formed with low concentrations of fibrinogen was high, while cell elongation was significantly reduced in hydrogels formed with high concentrations of fibrinogen. As presented in [Figure 5B], the cell elongation length was highest in fibrin hydrogels with a loose structure. The cell elongation lengths of stem cells in fibrin hydrogels prepared with four concentrations of fibrinogen (1.25, 2.5, 5.0, and 10.0 mg / ml) were 112.5, 75.0, 25.2, and 7.4 μm, respectively, confirming a significantly higher microfiber length in hydrogels with a loose structure, and confirming that physical cell elongation can be controlled depending on the structural and physical properties of the hydrogel.

[0166]

[0167] Example 6: Activation of stem cell signaling according to hydrogel nanostructure

[0168] The interaction between hydrogel physical and structural properties and stem cells was evaluated by 3D culture of myocardial stem cells (MyoCSCs).

[0169] Stem cell-incorporated hydrogels were prepared using the same method as in <Example 5> above, and then fixed with 4% formaldehyde for 2 hours. After washing three times with PBS, they were reacted with β-integrin (β-integrin, Cell Signaling Technology) and p-FAK (cell signaling technology) primary antibodies at 37°C for 1 hour, and then reacted with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) for 30 minutes.

[0170] Nuclei were stained with DAPI (diamidino-2-phenylindole, Molecular Probes), and the expression levels of β-integrin and p-FAK were evaluated using a confocal microscope. Western blotting was performed to evaluate integrin-mediated cell trafficking pathways. Intracellular protein secretion was extracted from 3D hydrogels or 2D culture environments and then subjected to PAGE electrophoresis. The PAGE gel was blotted onto an Immobilon (Sigma) membrane and incubated with primary antibodies against p-FAK (Cell Signaling Technology), p-JNK (Cell Signaling Technology), and p-β-catenin (Cell Signaling Technology) for 1 hour, followed by incubation with AP-conjugated secondary antibodies for 30 minutes. Afterwards, the membrane was reacted with ECL (ThermoFisher Scientific) substrate, and band intensities were measured. The results were compared after being corrected for the intensity of β-actin expression.

[0171]

[0172] As shown in [Figure 6], the expression of β-integrin, p-FAK, and β-catenin of stem cells incorporated into a 3D environment differed depending on the structural characteristics of the hydrogel. Compared to the 2D culture environment, the expression of p-FAK, p-JNK, and β-catenin in the 3D culture environment significantly increased. In particular, the expression rate of p-FAK, p-JNK, and β-catenin increased in the sparse hydrogel structure in an inverse relationship with the concentration of the hydrogel constituent polymer.

[0173] In the case of P-FAK, the expression increased by 85.4, 67.8, 34.2, and 0.7 times in stem cells in hydrogels composed of fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg compared to the 2D culture environment. In the case of P-JNK, the expression increased by 35.4, 25.4, 12.6, and 0.8 times in stem cells in hydrogels composed of fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg compared to the 2D culture environment, and in the case of p-β-catenin, the expression rate increased by 12.5, 9.4, 3.5, and 0.9 times.

[0174] The above results confirmed that microfiber formation increases with the degree of cell elongation, and that the expression of p-FAK, p-JNK, and β-catenin also increases, confirming that cell elongation can activate cell signaling pathways.

[0175]

[0176] Example 7: Characterization of Wnt signaling pathway mRNA expression in stem cells in 2D and 3D culture environments.

[0177] In the present invention, the expression rate of mRNA related to the Wnt signaling pathway of stem cells in a 2D culture or 3D hydrogel culture environment is measured using a PCR microarray kit (RT 2 The results were evaluated using the Profiler PCR Array (Qiagen).

[0178] Total RNA was extracted from MyoCSCs cultured in a 2D environment using TRIzol (Invitrogen), and cDNA was synthesized using the reverse transcriptase provided in the kit.

[0179] 3D hydrogel was prepared with 1.25 mg / ml fibrinogen and 0.5 unit / ml thrombin solution, and 1.0×10 6 MyoCSCs were cultured. After 3 days of culture, total RNA and cDNA were synthesized using the same method as in the 2D culture environment.

[0180] The expression rates of p-FAK, p-JNK, and p-β-catenin were confirmed. RT 2 The expression of 82 genes, including Wnt ligands, receptors, and downstream target genes, was assessed using the Profiler PCR Array (Qiagen). The expression rate was expressed as a percentage using the ΔΔCT method, adjusted for housekeeping genes, compared to MyoCSCs in a 2D culture environment.

[0181]

[0182] As shown in [Figure 8], the expression of Wnt ligands, receptors, and Wnt target downstream mRNAs was significantly increased in a 3D hydrogel environment. The Wnt signaling pathway is activated by extracellular physical stimuli, and in particular, increases with the degree of microfilament formation of actin filaments within the cytoplasm.

[0183] As presented in this example, it was confirmed at the mRNA level that physical stimulation of stem cells was induced in a 3D hydrogel culture environment, resulting in the promotion of actin filament organization, a significant increase in microfiber formation as a result of cell elongation, and the activation of the final Wnt signaling pathway.

[0184] Compared to the 2D culture environment, the expression rate of Wnt ligand mRNA increased 56.6-fold for WNT1, 32.3-fold for WNT10A, 15.4-fold for WNT10B, 17.0-fold for WNT11, 77.6-fold for WNT2B, 27.5-fold for WNT3A, and 47.0-fold for WNT6 (p < 0.05). Wnt receptor mRNA was also expressed upregulated in stem cells in the 3D culture environment.

[0185] In a 3D environment, stem cell mRNA expression was increased 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 compared to the 2D culture environment. As a result, significant changes in the expression of target genes affected by the downstream Wnt signaling pathway were also confirmed.

[0186] Compared to the 2D culture environment, in stem cells in the 3D culture environment, CCND1 increased 12.2-fold, CCND2 7.5-fold, CD44 3.0-fold, CDX1 90.2-fold, CDX2 63.1-fold, FGF4 47.7-fold, FGF9 62.5-fold, FN1 6.7-fold, MYC 29.8-fold, MYCN 45.0-fold, NANOG 49.3-fold, SOX2 84.1-fold, and VEGFA 87.9-fold. These results suggest that adjusting the 3D hydrogel structure can activate the Wnt signaling pathway of stem cells and, as a result, increase functional gene expression.

[0187]

[0188] Example 8: Exosome production in stem cells according to 3D hydrogel nanostructures and cell elongation.

[0189] The relationship between stem cell elongation, p-FAK, and exosome production according to hydrogel structural characteristics was evaluated.

[0190]

[0191] Stem cell-incorporated hydrogels were prepared using the same method as in <Example 5> above, and then fixed for 2 hours by adding 4% formaldehyde. After washing three times with PBS, they were reacted with p-FAK (Cell Signaling Technology) primary antibody at 37°C for 1 hour, and then reacted with Alexa Fluor 488-conugated anti-mouse IgG (Invitrogen) for 30 minutes. Exosome markers, anti-CD9, CD63, and CD81, were reacted with markers (Thermo Fisher Scientific) for 1 hour, and then reacted with Alexa Fluor 594 (Molecular Probes), and the nucleus was stained with DAPI (diamidino-2-phenylindole, Molecular Probes). p-FAK expression and the rate of exosome production in the cytoplasm were evaluated using a confocal microscope.

[0192]

[0193] Intracellular exosome production according to fibrinogen concentrationFibrinogen (mg / ml)CD9CD63CD811.25 ㎎13251543.11845.22.5 ㎎758689.1714.55 ㎎214184.5245.110 ㎎102112.3156.3

[0194] As presented in [Figure 9], the expression level of p-FAK and exosome production of stem cells in the 3D hydrogel increased proportionally. p-FAK expression increased in proportion to cell elongation, and particles positive for exosome markers CD9, CD63, and CD81 in the cytoplasm significantly increased according to the level of p-FAK expression. As in the previous results, the intensity of exosome-positive particles significantly increased in cells in the loose hydrogel structure manufactured with low-concentration fibrinogen. The above results confirmed that p-FAK expression can be controlled depending on the hydrogel structure, and as a result, exosome production of stem cells can be controlled.

[0195]

[0196] Example 9: Exosome secretion into cell culture media according to 3D hydrogel nanostructures

[0197] The relationship between cell elongation of cardiac stem cells and exosome secretion rate into cell culture medium according to hydrogel structural characteristics was evaluated.

[0198]

[0199] One ml of fibrinogen solutions at four concentrations (1.25, 2.5, 5.0, and 10.0 mg / ml) were mixed with one million MyoCSCs, and then each was mixed with the same volume of thrombin solution. Fibrin hydrogels containing MyoCSCs were prepared through polymerization and cross-linking reactions at 37°C for 1 hour. A serum-free medium containing 50 μg / ml ascorbic acid and 100 μg / ml N-acetyl cysteine ​​in DMEM / F12 was prepared as a cell culture solution, and the prepared cell culture solution was added to the fibrin containing MyoCSCs and cultured for 24 hours.

[0200] Exosomes were isolated from cell culture fluid collected 24 hours later using the tangential flow filtration (TFF) method, using a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The number of nanoparticles in the culture fluid was evaluated using a nanoparticle tracking analyzer (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical) for the concentrated and purified exosomes. The protein content in the exosomes was measured using microBCA (Fisher Thermo Scientific). Exosomes were isolated from the cell culture fluid obtained after culturing the same MyoCSCs in a 2D culture environment for 24 hours.

[0201]

[0202] Exosome production yield in 3D culture environment according to the polymer content of 3D hydrogel composition Fibrinogen (mg / ml) Number of exosomes (x10 9 / ㎖)Protein(㎍ / ㎖)1.2525.4318.52.516.1213.155.768.4101.118.52D1.212.1

[0203] As presented in [Figure 10], a significant difference was observed in the number of exosomes produced and secreted from stem cells 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 hydrogels with a loose structure manufactured at a low polymer concentration. The number of exosomes in fibrin hydrogels manufactured at four fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml was 25.4 × 10 9 / ㎖, 16.1 x 10 9 / ㎖, 5.7 x 10 9 / ㎖, 1.1 x 10 9 The number of exosomes was significantly higher in the fibrin hydrogels with a loose structure prepared from low concentrations of fibrinogen (100 μg / mL).

[0204] The protein content was also significantly higher in the stem cells secreted within the hydrogels with a loose structure, similar to the number of exosomes. The protein contents in the fibrin hydrogels prepared according to the fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml were 318.5, 213.1, 68.4, and 18.5 μg / ml, respectively, indicating that the protein content produced and isolated from stem cells in the cell culture medium in the fibrin hydrogels with a loose structure was significantly higher. It was confirmed that stem cells cultured within the fibrin hydrogels except for the 10.0 mg / ml concentration could support significantly higher exosome secretion compared to stem cells cultured in a 2D culture environment.

[0205]

[0206] Example 10: Confirmation of the size of exosomes produced in 2D culture and 3D hydrogel culture environments.

[0207] In the present invention, the structural differences of exosomes produced depending on the culture environment were tested.

[0208] Myocardial stem cells were cultured by encapsulating them in a 3D fibrin hydrogel according to the method presented in <Example 9> above, and then exosomes were isolated.

[0209] The size of nanoparticles in the culture medium was measured using a nanoparticle tracking analyzer (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical) for the concentrated and purified exosomes. Exosomes were isolated from the cell culture medium obtained after culturing the same MyoCSCs in a 2D culture environment for 24 hours, and the size of the nanoparticles was measured using the same method.

[0210]

[0211] As presented in [Figure 11], no difference in exosome particle size was observed according to the culture environment. The average diameters of exosomes generated in gels composed of fibrinogen concentrations of 1.25, 2.5, 5.0, and 10.0 mg / ml were 118, 116, 117, and 121 nm, respectively, which were no different from the average diameter of 121 nm of exosomes generated in a 2D environment. The Mode value, which is the size of the most frequently occurring nanoparticle, also did not differ according to the 2D and 3D culture environments, and no difference according to the concentration of the hydrogel constituent polymer could be confirmed.

[0212]

[0213] Example 11: Immunophenotypic Characterization of Secreted Exosomes Generated from Stem Cells in 2D and 3D Hydrogel Culture Environments

[0214] The immunophenotypic characteristics of exosomes generated and isolated from cardiac stem cells in 2D and 3D culture environments were analyzed using a multiplex bead kit (MACS Plex Exosome Kit, Mitenyl Biotec).

[0215] Myocardial stem cells were cultured by encapsulating them in a 3D fibrin hydrogel according to the method presented in <Example 9> above, and then exosomes were isolated.

[0216] Exosomes were analyzed by centrifuging at 2,500 × g for 15 minutes and collecting the supernatant. 30 μl of exosomes were mixed 1:1 with MACSPlex buffer. 8 μl of MACSPlex exosome capture beads were added to each well of a MACSPlex 96-well plate, followed by the addition of 60 μl of exosomes. After incubation at room temperature for 30 minutes, the immunohistochemical characteristics of exosomes were analyzed using a flow cytometer, and the expression level was analyzed by expression intensity (median APC-A).

[0217]

[0218] Immunophenotypic expression characteristics of myocardial stem cell exosomes according to culture environment. Marker3D2DCD3913.41089.4CD41182.5244.4CD19222.6-765.5CD8486.4579.8HLA-DR / DP / DQ1082.81668.5CD561774.61190.8CD105137190.5266637.3CD2561.7726.8CD1c9 02.7-112.3CD251439629.3CD49e65388.369071ROR16527.37569CD209858.51038.1CD9 144216.3197336.4SSEA-45019.96944HLA-ABC15762278.3CD63507739.42686936.3CD40 1852.3980.9CD62P305.8379.1CD11c66.8193.1CD81265857.1381481.1MCSP6091.8201 74.4CD1466276.77665.9CD41b1417.91087.5CD42a289.3434.5CD242143.12234.2CD869 821045.2CD44219282.9264876.4CD3265486.65488CD133 / 12256.93287.2CD29215551. 9277074.2CD69653.5-917.2CD1421115.7997.8CD451536.2846.6CD311817.61721.1REA Control766.7455.2CD20760.6188.3CD141854.92737.8mIgG1 Control25592017.3

[0219] As presented in [Table 4] and [Fig. 12], there was no difference in the immunophenotypic characteristics of exosomes produced depending on the culture environment. The expression intensities of exosome markers CD9, CD63, and CD81 were high in both 2D and 3D culture environments, but no differences were observed depending on the culture environment. The expression of cardiomyocyte-derived stem cell markers CD105, CD44, and CD29 also did not differ depending on the 2D and 3D culture environments, and the expression intensities were high in all exosomes.

[0220]

[0221] Example 12: Exosome production rate according to stem cell type in 2D and 3D culture environments

[0222] In the present invention, the yield of exosomes was analyzed according to the culture environment and stem cell type. Adipose, bone marrow, umbilical cord blood, synovial membrane, umbilical cord-derived mesenchymal stem cells, peripheral nerve-derived adult stem cells, and cardiac muscle-derived stem cells were cultured in 2D and 3D environments.

[0223] Each of the above stem cells was cultured by encapsulating them in a 3D fibrin hydrogel according to the method presented 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 (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical).

[0224]

[0225] As presented in [Figure 13], significant differences were observed in the number of exosomes produced depending on the culture environment. The number of exosomes produced in the 3D culture environment significantly increased by 1.7 to 2.9 times compared to the 2D culture environment. In all of the myocardium-derived stem cells (MyoCSCs), peripheral neural stem cells (PNSCs), umbilical cord blood (UC-MSCs), and bone marrow (BM-MSCs)-derived mesenchymal stem cells, a significant increase was confirmed in the 3D environment compared to the 2D culture environment.

[0226]

[0227] Example 13: Exosome production rate according to the number of cultured cells in a 3D fibrin hydrogel culture environment

[0228] In the present invention, the yield of exosomes was analyzed according to the culture density of stem cells.

[0229] 2, 10, 20, 200, 1,000, 2,000 x 10 per 1 ml of thrombin solution 4After mixing with cells, each was combined with a 2.5 mg / ml fibrinogen solution, and then cultured by incorporating into a 3D fibrin hydrogel according to the method presented in <Example 9>, and then exosomes were isolated.

[0230] The total number of exosomes was measured using a nanoparticle tracking analyzer (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical) after concentrating and purifying the exosomes.

[0231]

[0232] As presented in [Figure 14], the exosome production rate increased in proportion to the number of cells incorporated into the hydrogel. When 10,000, 50,000, 100,000, 1 million, 5 million, and 10 million cells were incorporated and cultured per ㎖ of hydrogel, the exosome production rate was 2.6, 4.3, 8.5, 25.4, 32.9, and 12.5 × 10 9 Dog exosomes were generated.

[0233] For fibrin hydrogel, up to 5 million cells per ml could be cultured, which corresponds to 3.3 x 10 cells per ml of cell culture medium. 10 Exosomes could be produced. When the number of cells exceeded 10 million per ml, the hydrogel contracted due to the cells, resulting in the hydrogel detaching from the culture vessel, compressing the cell-mediated hydrogel and reducing the exosome production rate.

[0234] For 2D culture environment, cm 2 The maximum number of cells that can be cultured per cell varies depending on the stem cell type, but 10,000 to 50,000 cells can be cultured, but in the case of a 3D culture environment, 10,000 to 50,000 cells can be cultured per cm 2 It was confirmed that this is a high-density culture method that can culture up to 10,000 to 5 million cells per unit area, enabling high-density culture, and that the number of exosomes that can be produced can also significantly increase as the number of cells cultured per unit area increases.

[0235]

[0236] Example 14: Exosome production rate according to 3D hydrogel polymer components

[0237] In this invention, we investigated the correlation between the microfiber production and exosome production rate of stem cells within hydrogels, depending on the type of hydrogel constituent polymer. Hydrogels containing fibrin, collagen, gelatin, hyaluronan, and chondroitin were applied as 3D cell culture matrices, and the exosome production rate was investigated.

[0238] 2 x 10 per 1 ml of crosslinking agent solution 6 After suspension with cells, 2.5 mg / ml of fibrinogen, 0.2% collagen, 0.4% gelatin, 0.2% hyaluronan, and 0.2% chondroitin solution were mixed in a 1:1 ratio, and then polymerization and cross-linking reaction was performed at 37°C for 1 hour to obtain 1 x 10 per ml of hydrogel. 6 Fibrin, collagen, gelatin, hyaluronan, and chondroitin hydrogels containing myocardial stem cells were prepared, respectively.

[0239] A serum-free medium containing 50 μg / mL ascorbic acid and 100 μg / mL N-acetyl cysteine ​​in DMEM / F12 was prepared as a cell culture solution, and the prepared cell culture solution was added to fibrin containing MyoCSCs and cultured for 24 hours.

[0240] Exosomes were isolated from cell culture fluid collected 24 hours later using the tangential flow filtration (TFF) method, using a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The total number of exosomes was measured using a nanoparticle tracking analyzer (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical) for the concentrated and purified exosomes.

[0241]

[0242] As presented in [Figure 15], the microfiber lengths of cardiac stem cells encapsulated in fibrin, collagen, gelatin, hyaluronan, and chondroitin hydrogels were 112.5, 78.5, 63.4, 58.9, and 61.8 μm, respectively, with the fibrin hydrogel being the highest. The microfiber length of cardiac stem cells within the hydrogels showed a proportional correlation with the exosome production rate.

[0243] Exosome production by cardiac stem cells in fibrin, collagen, gelatin, hyaluronan, and chondroitin hydrogels was 25.4, 12.7, 8.5, 6.4, and 7.2 × 10 9 Exosome production rates were significantly higher in myocardial stem cells within fibrin and collagen hydrogels.

[0244] The above results show that the cell elongation of stem cells embedded in hydrogels varies depending on the hydrogel polymer component, and that the length of microfibers reflecting cell elongation is proportionally related to the exosome production rate.

[0245]

[0246] Example 15: Exosome production rate in a 3D synthetic polymer and copolymer hydrogel culture environment

[0247] In the present invention, a 3D culture environment was constructed using a hydrogel composed of a synthetic polymer component and a synthetic polymer and fibrin copolymer hydrogel, and then myocardial stem cells were cultured and the exosome production rate according to the hydrogel type was analyzed.

[0248]

[0249] 2 x 10 per 1 ml of thrombin or cross-linking agent solution 6 After mixing with cells and 1:1 with 2.5 mg / ml fibrinogen solution or 5% PGA, PEG, PLGA PLLA polymer solution, polymerization and cross-linking reaction was performed at 37°C for 1 hour to obtain 1 x 10 per ml of hydrogel. 6Fibrin and PGA, PEG, PLGA, and PLLA hydrogels containing myocardial stem cells were prepared.

[0250] To enhance the cytocompatibility of synthetic polymer hydrogels, a hydrogel copolymerized with fibrin was prepared and used as a 3D matrix. To infiltrate cardiac stem cells into the fibrin and synthetic polymer copolymer hydrogel, a 2 × 10 6 Myocardial stem cells were mixed with a solution containing a cross-linking agent and thrombin, and then mixed 1:1 with 5% PGA, PEG, PLGA PLLA polymer solution containing 2.5 mg / mL fibrinogen solution, and then polymerized and cross-linked at 37°C for 1 hour to obtain 1 x 10 per mL. 6 Fibrin containing myocardial stem cells was incorporated into a hydrogel copolymerized with PGA, PEG, PLGA, and PLLA.

[0251] A serum-free medium containing 50 μg / mL ascorbic acid and 100 μg / mL N-acetyl cysteine ​​in DMEM / F12 was prepared as a cell culture solution, and the prepared cell culture solution was added to fibrin containing MyoCSCs and cultured for 24 hours.

[0252] Exosomes were isolated from cell culture fluid collected 24 hours later using the tangential flow filtration (TFF) method, using a cartridge (Repligen) with a molecular weight cut-off of 300 kDa. The total number of exosomes was measured using a nanoparticle tracking analyzer (Nanoparticle Tracking Analyzer, NanoSight Pro, Malvern Panalytical) for the concentrated and purified exosomes.

[0253]

[0254] As presented in [Figure 16], the degree of microfiber formation of cardiac stem cells in the synthetic polymer hydrogel was 1 / 10 compared to that in the fibrin hydrogel, and the microfiber lengths of cardiac stem cells in the hydrogels containing PGA, PEG, PLGA, and PLLA components were 13.5, 12.7, 14.6, and 13.7 ㎛, respectively, confirming low microfiber formation.

[0255] However, in the case of hydrogels mixed with synthetic polymers and fibrin hydrogels, the lengths of myocardial stem cell microfibers were 42.5, 58.9, 85.4, and 65.8 µm, indicating that cell compatibility was improved through fibrin mixing, and the degree of microfiber formation, which indicates cell elongation, significantly increased as the ligand that bonds with the hydrogel matrix and cells increased.

[0256] The exosome production rate of myocardial stem cells in hydrogels containing synthetic polymers or synthetic polymers mixed with fibrin was significantly enhanced in hydrogels containing synthetic polymers mixed with fibrin. In the case of 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 It showed a low production rate.

[0257] In the case of synthetic polymer hydrogels mixed with fibrin, the exosome production rate increased, and when PGA, PEG, PLGA, and PLLA polymer hydrogels mixed with fibrin were cultured as 3D substrates, the exosome production rates were 18.5, 19.7, 16.7, and 12.8 × 10 9 It showed a significant increase compared to the synthetic polymer hydrogel before mixing.

[0258] The above results indicate that the formation of microfibers in cells is related to ligands that can bind to cells within the hydrogel, and that the degree of microfiber formation is closely related to the rate of exosome production from stem cells. In the case of synthetic polymer hydrogels, it can be confirmed that cell compatibility is low, resulting in a decrease in microfiber production and exosome production rates.

[0259]

[0260] Example 16: miRNA Expression in Stem Cell-Derived Exosomes Generated in 2D and 3D Hydrogel Culture Environments

[0261] 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 action mediators of exosomes, was measured.

[0262] Myocardial stem cells were cultured by encapsulating them in 3D fibrin hydrogels according to the method presented in <Example 9>, and then exosomes were isolated. The exosomes were centrifuged at 2,500 xg for 15 minutes, and miRNA was isolated from the supernatant using a miRNA isolation kit (miRNA isolation kit, mirvana miRNA isolation Kit, Invitrogen). The miRNA extracted from the exosomes was used to prepare a miRNA library through a reverse transcriptase reaction (Taqman MicroRNA Reserve Transcription Kit, Invitrogen). Quantitative PCR (TaqMan MicroRNA Assay Kit) was performed using miRNA-specific target probes (Affymatrix) using the Taqman method.

[0263]

[0264] As presented in [Fig. 16], it was confirmed that the content of let-7b-3b, miR126-5p, miR145-5p, miR146a-5p, and miR185-5p, which are miRNAs that act on angiogenesis, tissue regeneration, and inflammation control, was significantly increased in exosomes produced in the 3D hydrogel culture environment of the present invention compared to exosomes obtained in a 2D culture environment. In other words, it was confirmed that not only can exosome production be increased through the 3D hydrogel culture of the present invention, but also the composition of effective ingredients in exosomes can be increased, thereby enhancing angiogenesis, tissue regeneration, and inflammation control efficacy.

[0265]

[0266] Example 17: Wound repair, angiogenesis, and anti-inflammatory effects of stem cell-derived exosomes produced in 2D and 3D hydrogel culture environments.

[0267] In order to analyze the difference in efficacy of stem cell-derived exosomes produced according to the culture environment in the present invention, the effects on the growth of fibroblasts involved in wound repair, the effects on the growth of vascular endothelial cells involved in angiogenesis and the elongation of blood vessels, and the effects on the separation of inflammatory cytokines secreted from inflammatory cells were tested.

[0268] Each of adipose, bone marrow, and muscle-derived mesenchymal stem cell, peripheral nerve-derived stem cell, and cardiac muscle-derived stem cell was cultured by entrapping them in a 3D fibrin hydrogel according to the method presented in <Example 9>, and then exosomes were isolated.

[0269]

[0270] The efficacy of exosomes was evaluated in a cell-based manner using fibroblasts (3T3-L1), human umbilical cord vein endothelial cells (HUVECs), and RAW264.7 inflammatory cells. The ability of exosomes to induce cell growth was tested by seeding DFs and HUVECs cells at 5,000 cells per well in 96-well plates, adding 1.0E+08 exosomes to each well, and incubating for 24 hours. After incubation, the cells were lysed in 0.1% SDS / TE solution, and the growth rate was analyzed as a ratio (fold) compared to cells that were not administered exosomes using the PicoGreen dsDNA quantitation kit.

[0271] To evaluate the effect of exosomes on vascular tube growth, microspheres composed of 300 HUVEC cells were prepared, and then the microspheres were embedded in a collagen gel and cultured. The length of the vascular tubes increased depending on the addition of exosomes, and the effect of exosomes on vascular tube growth was evaluated.

[0272] One hundred thousand RAW264.7 inflammatory cells were seeded in a 24-well plate and sensitized with 10 μg LPS (lipopolysaccharide, Sigma). Exosomes were added during sensitization with LPS, and the concentrations of TNF-α and IL-β secreted from inflammatory cells by LPS sensitization were analyzed using an ELISA kit (R&D Systems).

[0273]

[0274] As a result of treating 3T3-L1 with the exosomes generated above, as shown in [Fig. 18], it was confirmed that 3T3-L1 cell growth was significantly increased by the stem cell-derived exosomes produced in the 3D hydrogel culture environment of the present invention compared to the 2D culture environment. When the stem cell exosomes derived from fat, bone marrow, myocardium, peripheral nerve, and muscle generated in the 3D culture environment were treated, cell growth increased by 28%, 31%, 119%, 28%, and 36% compared to the exosomes generated in the 2D culture environment, confirming that the wound repair effect was high.

[0275] As a result of treating HUVEC cells with exosomes produced in a 3D culture environment, as shown in [Figure 19], the exosomes produced in a 3D culture environment showed a significantly higher effect in inducing HUVEC cell growth. When treated with stem cell exosomes derived from adipose, bone marrow, cardiac muscle, peripheral nerve, and muscle produced in a 3D culture environment, they showed an effect in inducing HUVEC growth of 10%, 18%, 56%, 50%, and 38%, respectively, compared to the exosomes produced in a 2D culture environment.

[0276] The degree of growth of vascular tubes formed in HUVECs treated with exosomes generated in a 3D culture environment is shown in [Figure 20]. The lengths of HUVEC vascular tubes formed with exosomes derived from adipose, bone marrow, myocardium, peripheral nerve, and muscle stem cells in a 3D culture environment were 3.1, 2.8, 4.5, 3.5, and 4.9 mm, respectively, while those of exosomes generated in a 2D culture environment were 2.7, 2.3, 3.8, 2.8, and 3.5 mm, respectively, confirming a significantly higher ability to induce vascular tube growth in exosomes generated in a 3D culture environment. The results of stem cell exosomes generated in a 3D culture environment promoting the growth of vascular endothelial cells and vascular tube growth are predicted to have high angiogenic efficacy.

[0277] As shown in [Figure 21] and [Figure 22], when exosomes produced in a 3D culture environment were treated on 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.

[0278] A significant decrease in the amount of TNF-α secretion was confirmed in exosomes generated in a 3D culture environment, and when exosomes from stem cells derived from bone marrow, adipose, cardiac, peripheral nerve, and muscle were treated, the levels were 302, 364, 213, 306, and 254 pg / ㎖, which were lower than when exosomes generated in a 2D culture environment were treated.

[0279] The IL-1β secretion inhibitory ability was also high when treated with exosomes generated in a 3D culture environment, and when treated with stem cell exosomes derived from bone marrow, fat, myocardium, peripheral nerve, and muscle, RAW264.7 cells secreted 330, 365, 245, 305, and 287 pg / ㎖, but when treated with stem cell exosomes generated in a 2D culture environment, the IL-1β secretion inhibitory ability was also high when treated with exosomes generated in a 3D culture environment, at 405, 421, 356, 364, and 358.

[0280] Based on the above cells, the wound repair, angiogenesis, and anti-inflammatory effects were significantly higher in stem cell exosomes in a 3D culture environment, confirming that the exosome effect can be increased depending on the culture environment.

[0281]

[0282] It was confirmed that exosomes produced using the method of the present invention increase miRNA components capable of regulating angiogenesis, tissue regeneration, and inflammation. Therefore, exosomes produced using the method of the present invention can be usefully utilized as compositions for angiogenesis, tissue regeneration, or anti-inflammatory purposes.

Claims

1. (a) a step of mixing a hydrogel in a sol state and stem cells, the hydrogel having a ligand capable of binding to the integrin of the cell and composed of a polymer chain capable of a sol-gel phase transition; and (b) A method for increasing the production of stem cell-derived exosomes by delivering physical stimulation through cell stretching, comprising the step of manufacturing a 3D hydrogel in which stem cells are uniformly captured in a three-dimensional manner by phase-transforming the hydrogel into a gel state and culturing stem cells.

2. In paragraph 1, The above method is a method for increasing the production of stem cell-derived exosomes by delivering a physical stimulus through cell stretching, characterized in that the method stretches the cells by directly binding the ligand of the hydrogel to the cells without a physical device.

3. In paragraph 1, A hydrogel having a ligand capable of binding to the above integrin, A natural polymer selected from the group consisting of collagen, fibrin, gelatin, hyaluronan, and chondroitin; or A method for increasing the production of stem cell-derived exosomes by transmitting physical stimulation through cell stretching, characterized in that the hydrogel is a copolymer of the above natural polymer material and a synthetic polymer material selected from the group consisting of PEG (Polyethylene Glycol), PLA (Polylactic Acid), PLGA (Polylactic Glycolic Acid), and PGA (Polyglycolic Acid).

4. In paragraph 1, A method for increasing the production of stem cell-derived exosomes by delivering physical stimulation through cell stretching, characterized in that the step (b) above adds a cross-linking agent to the mixture of step (a) to cause a phase transition of the hydrogel into a gel state.

5. In paragraph 1, A method for increasing the production of stem cell-derived exosomes by transmitting physical stimulation through cell stretching, characterized in that at least one polymer material selected from the group consisting of PEG, PLGA, PGA, chitosan, gelatin, collagen, chondroitin, and hyaluronic acid is additionally mixed in step (a) to control the physical strength of the 3D hydrogel in step (b).

6. In paragraph 1, A method for increasing the production of stem cell-derived exosomes by delivering a physical stimulus through cell stretching, characterized in that in order to enhance stability by inhibiting degradation of the 3D hydrogel by cells in the step (b), at least one matrix metalloproteinase (MMP) inhibitor selected from the group consisting of marimastat, batimastat, ilomastat (GM6001), and cipemastat capable of inhibiting at least one MMP selected from the group consisting of MMP-2, MMP-3, MMP-8, and MMP-9 is additionally mixed in the step (a).

7. In paragraph 1, A method for increasing the production of stem cell-derived exosomes by delivering physical stimulation through cell stretching, characterized in that in order to enhance stability by inhibiting cell-induced degradation of the 3D hydrogel in the step (b), at least one antifibrinolytic selected from the group consisting of aminocaproic acid and tranexamic acid is additionally mixed in the step (a).

8. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, characterized in that the 3D hydrogel of step (b) has a porous structure with a size of 50 nm to 300 nm, thereby providing cell-cell binding and having structural properties that promote cell stretching.

9. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, characterized in that the stem cells cultured in the step (b) above have an appearance of being stretched in a three-dimensional (3D) axial plane and the cells can be stretched to a length of 15 ㎛ to 400 ㎛.

10. In paragraph 1, 1.0×10 per ㎖ of hydrogel 4 ~ 5.0x10 6 A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, characterized in that stem cells can be cultured by incorporating them at a high density.

11. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, characterized in that the cultured stem cells are characterized in that the integrin-FAK-JNK-β-catenin signaling pathway is activated through the control of the physicochemical properties and structural characteristics of the hydrogel in the step (b).

12. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, wherein the cultured stem cells are characterized by activation of the Wnt signaling pathway and increased expression of Wnt signaling downstream target genes through regulation of hydrogel properties and structural characteristics in the step (b).

13. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, wherein the stem cell-derived exosomes produced by the above method are characterized by an increase in the composition of at least one angiogenesis-regulating miRNA selected from the group consisting of let-7b-3b and miR126.

14. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, wherein the stem cell-derived exosomes produced by the above method are characterized by an increase in the composition of at least one inflammation-regulating miRNA selected from the group consisting of miR145-5p, miR146a-5p, and miR185-5p.

15. In paragraph 1, A method for producing stem cell-derived exosomes through physical stimulation via cell stretching, characterized in that the stem cells may be adult stem cells or mesenchymal stem cells, and are preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, myocardium-derived stem cells, muscle-derived stem cells, peripheral nerve-derived stem cells, synovium-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells.

16. A composition for angiogenesis, tissue regeneration, or anti-inflammation, comprising a stem cell-derived exosome prepared by any one of the methods of claims 1 to 15 as an effective ingredient.

17. In paragraph 16, A composition characterized in that the composition is a pharmaceutical composition, a quasi-drug composition or a cosmetic composition.

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