Methods, devices and cells for inhibiting, delaying or reversing cellular senescence

Abstract

The present invention relates to a method, apparatus, and cell for inhibiting, delaying, or reversing cellular senescence. The method includes the following steps: flowing isolated cells through a microchannel; and impinging the flowing cells into an impact surface mounted on the flow path of the cells to apply a physical impact to the cells, resulting in the inhibition, delay, or reversal of cellular senescence while maintaining high biological activity. Specifically, the present invention relates to a method for further increasing the value of stem cells as therapeutic cells for various degenerative diseases by maintaining the undifferentiated state of stem cells even during long-term culture periods to maintain pluripotency, and to cells obtained by the method.

Description

[0001] This application claims the priority and benefits of Korean Patent Applications No. 10-2020-0094616 and No. 10-2021-0098400, filed on July 29, 2020 and July 27, 2021, respectively, with the Korean Intellectual Property Office, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for inhibiting, delaying or reversing cell senescence by applying an impact to cells in flow using a microfluidic system, a microfluidic system, and cells in which senescence is inhibited, delayed or reversed obtained from the method or the microfluidic system. Background Art

[0003] The cytoskeleton induces various intracellular changes through the polymerization / depolymerization of actin in response to extracellular stimuli. These changes regulate cell migration and invasion and affect the activity of ion channels present in cells, ultimately leading to cell survival, proliferation, death, motility, and secretion through various intracellular signaling pathways.

[0004] Cell senescence refers to an irreversible biological phenomenon in which the cell cycle of cells exposed to various physical, chemical, and biological stresses slows down and cell division decreases or permanently stops. As senescence progresses, cell division not only decreases or stops, but cell damage accumulates due to the unstable transmission of genetic information. The causes of senescence related to this genetic information include genomic instability, telomere loss, and epigenetic changes.

[0005] However, senescence at the cellular level is a complex process that cannot be effectively controlled by single-factor regulation. No method has been developed for effectively inhibiting or delaying cell senescence or reversibly restoring senescent cells to normal cells to have a phenotype similar to that of young cells.

[0006] Meanwhile, mesenchymal stem cells (MSCs), which are first recognized in the bone marrow, are pluripotent cells with great potential in regenerative medicine. Mesenchymal stem cells can differentiate into several types of mesenchymal lineages such as osteocytes, chondrocytes, adipocytes, myocytes, and fibroblasts. Because mesenchymal stem cells have immunomodulatory activity, they can be used as compositions for treating various autoimmune and inflammatory diseases such as transplantation promoters, fetal grafts, and host diseases. Specifically, due to the excellent therapeutic efficacy of mesenchymal stem cells, the demand for regenerative medicine continues to increase. However, during in vitro expansion to ensure a therapeutically effective amount of cells, the longer the culture period, the less cell proliferation, and the undifferentiated state cannot be maintained, thus losing the regenerative ability and pluripotency unique to stem cells. Therefore, if a process for inhibiting stem cell aging or inducing reverse aging is developed, the therapeutic effectiveness of stem cells can be significantly improved.

[0007] Throughout the specification, many papers and patent documents are cited, and their citations are indicated. The disclosures of the cited papers and patent documents are hereby incorporated by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

[0008] "Prior art documents"

[0009] "Patent references"

[0010] (Patent reference 0001) Patent reference 1, Korean Application No. 2015-0167693 Summary of the Invention

[0011] The present invention provides a method for inhibiting, delaying, or reversing cell aging and cells in which aging is inhibited, delayed, or reversed using the method.

[0012] The present invention also provides a method for maintaining or enhancing the pluripotency of stem cells.

[0013] The present invention also provides an apparatus for inhibiting, delaying, or reversing the aging of isolated cells.

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description, claims, and drawings of the invention.

[0015] According to an embodiment of the present invention, the present invention provides a method for inhibiting, delaying, or reversing cell aging, the method comprising the steps of:

[0016] Causing isolated cells to flow; and

[0017] Cells are impacted into an impact surface installed in the flow path of the cells to apply a physical impact to the cells.

[0018] The inventors of the present invention made in - depth research efforts to develop a method for delaying the senescence of various therapeutic and research cells, including stem cells, and maintaining a high level of biological activity (such as survival rate, proliferation rate, and differentiation ability) for a long time. As a result, the inventors found that in an in - vitro environment including a flow channel and an impact surface (i.e., in a microfluidic system), by allowing target cells to flow with certain parameters and impacting the cells into the impact surface to apply a physical impact to the cells, reorganization of the cytoskeleton can be induced, thereby significantly delaying, reversing, or inhibiting cell senescence. Specifically, the inventors have compared various senescence factors of cells that have passed through the microfluidic system as described above (such as cell size, the level of intracellular reactive oxygen species, and the expression levels of senescence - related proteins or genes) with various senescence factors of control cells to which the above - described microfluidic system has not been applied, to confirm the senescence - inhibiting effect of the cells according to the present invention, and completed the present invention.

[0019] As used herein, the term "flow" includes the flow of a fluid containing cells.

[0020] As used herein, the term "impact surface" refers to a surface into which cells in flow impact by being installed on the flow path while forming a predetermined angle with the flow path of the cells. The angle between the axis of the flow path and the impact surface can be about less than 180 degrees, for example, about 10 degrees to about 170 degrees, about 20 degrees to about 170 degrees, about 20 degrees to about 160 degrees, about 30 degrees to about 160 degrees, about 30 degrees to about 150 degrees, about 40 degrees to about 150 degrees, about 40 degrees to about 140 degrees, about 50 degrees to about 140 degrees, about 50 degrees to about 135 degrees, about 50 degrees to about 130 degrees, about 60 degrees to about 130 degrees, about 60 degrees to about 120 degrees, about 70 degrees to about 120 degrees, about 70 degrees to about 110 degrees, about 80 degrees to about 110 degrees, about 80 degrees to about 100 degrees, about 85 degrees to about 100 degrees, about 85 degrees to about 95 degrees, or about 90 degrees, but is not limited thereto. Any angle that can collide with cells moving in the axial direction of the flow path by forming a predetermined angle and not being parallel to the axis of the flow path can be applied without limitation.

[0021] According to an embodiment, the flow path can be, for example, a T - shape in which cells flow in a straight line and then diverge 90 degrees to the left and right from the flow direction at a specific point. In this case, the impact surface becomes the surface of the junction that is at a perpendicular angle to the axis of the flow path. The impact surface can have a flat shape or can include an uneven structure containing recesses, protrusions, or a combination thereof for effective cell impact and cytoskeleton reorganization.

[0022] In an embodiment, flow can be performed by injecting a fluid containing cells into a microchannel in a microfluidic system according to an embodiment of the present invention to cause it to flow.

[0023] As used herein, the term "microchannel" refers to a migration path of a fluid having a hydraulic diameter of less than about 1 mm or a flow rate of the fluid at the micro level (μL) per minute. For example, the flow rate of the fluid can be about several tens of μL to several thousand μL per minute, such as about several tens of μL to several hundred μL per minute or about several hundred μL to several thousand μL per minute, and is not limited thereto.

[0024] A microfluidic system according to an embodiment of the present invention includes: an inlet for injecting cells or a fluid; an outlet for discharging cells; a microchannel connecting the inlet and the outlet to allow the fluid to flow; and a control unit for controlling the flow rate of the microchannel.

[0025] According to an embodiment, flow can be performed according to predetermined parameters such that reorganization of the cytoskeleton occurs without physical shock that causes cell death.

[0026] As used herein, the term "reorganization of the cytoskeleton" refers to a series of continuous processes in which tubulin that constitutes microtubules as cytoskeletal fibers depolymerizes by physical shock and then polymerizes again, resulting in denaturation and recovery of the cytoskeleton. The present inventors found that during this cytoskeleton reorganization process, the density of actin, which is a monomer component of microfilaments as another component of the cytoskeleton, increases and the structure becomes dense, and as a result, the characteristic phenotype of cells without senescence is reproduced.

[0027] The inventors found that when a physical shock of appropriate energy is applied to cells (e.g., senescent cells) to the extent that denaturation and recovery of the cytoskeleton continuously occur without completely destroying the cells, senescence-related phenotypes (such as, for example, cell size, levels of reactive oxygen species, expression levels of senescence-related proteins or genes, and actin density) have changed to substantially suppress, delay, or induce reversal of senescence.

[0028] Therefore, the "predetermined parameters" refer to a series of hydrodynamic variables that can affect the amount of energy applied when cells collide with an impact surface, such as, for example, the flow rate, density, or viscosity of the fluid containing cells and the diameter of the microchannel.

[0029] Specifically, the parameters can be determined by one or more factors selected from the density, flow rate, characteristic length, and viscosity coefficient of the fluid containing cells.

[0030] As used herein, the term "characteristic length" means the length that has the greatest influence on the flow and can be, for example, the diameter of the microchannel.

[0031] More specifically, the parameter (Re) can be determined by Equation 1:

[0032] Re = ρVD / μ (Equation 1)

[0033] In Equation 1, ρ is the density of the fluid, V is the velocity of the fluid, D is the characteristic length of the fluid, and μ is the viscosity coefficient.

[0034] Equation 1 is an equation for deriving the Reynolds number. The Reynolds number is a dimensionless number that quantitatively represents the ratio of "inertial force" and "viscous force" in a fluid and can comprehensively define the flow conditions affected by various variables.

[0035] According to an embodiment of the present invention, the flow can be carried out under the condition that the parameter (Re) has a value less than 500. For example, "Re" can be about 1 to 500, about 10 to 500, about 10 to 450, about 10 to 400, about 10 to 350, about 10 to 300, about 10 to 290, about 20 to 290, about 30 to 290, about 40 to 290, about 50 to 290, about 60 to 290, about 70 to 290, about 80 to 290, about 90 to 290, about 100 to 290, about 100 to 280, about 110 to 280, about 120 to 280, about 130 to 280, about 130 to 270, about 170 to 260, about 180 to 260, about 190 to 260, about 200 to 260, about 200 to 255, about 205 to 255, about 210 to 255, about 210 to 250, about 220 to 250, about 225 to 250, about 230 to 250, about 235 to 250, about 235 to 248, or about 235 to 245, and is not limited thereto. In an example, the flow can be carried out with a non-viscous fluid, in which case "Re" can be as low as close to 0 (zero).

[0036] According to an embodiment of the present invention, the distance from the point where the cells start to flow in the microchannel to the impact surface can be from about 0.1 mm to about 50 mm. For example, the distance can be from about 0.1 mm to about 50 mm, from about 1 mm to about 50 mm, from about 0.1 mm to about 45 mm, from about 0.1 mm to about 40 mm, from about 1 mm to about 35 mm, from about 1 mm to about 30 mm, from about 1 mm to about 25 mm, from about 1 mm to about 20 mm, from about 2 mm to about 50 mm, from about 2 mm to about 40 mm, from about 2 mm to about 30 mm, from about 3 mm to about 50 mm, from about 3 mm to about 40 mm, from about 3 mm to about 30 mm, from about 3 mm to about 25 mm, from about 3 mm to about 20 mm, from about 3 mm to about 15 mm, from about 5 mm to about 50 mm, from about 5 mm to about 40 mm, from about 5 mm to about 30 mm, from about 5 mm to about 20 mm, from about 5 mm to about 15 mm, from about 5 mm to about 10 mm, from about 3 mm to about 10 mm, from about 3 mm to about 8 mm, or from about 0.1 mm to about 50 mm, and is not limited thereto.

[0037] The method of the present invention can be applied without limitation to any cell having a cytoskeleton. The cells can be, for example: fibroblasts; stem cells, including mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or combinations thereof; immune cells, including T cells, NK cells, B cells, dendritic cells, macrophages, or combinations thereof; precursor cells of fibroblasts, stem cells, or immune cells; or combinations thereof, and can be any cells for research and treatment.

[0038] In an embodiment, the cells can be stem cells or precursor cells. As used herein, the term "precursor cell" refers to a unipotent cell that is only partially differentiated and shares most phenotypes with stem cells.

[0039] As used herein, the term "stem cell" is an undifferentiated cell before differentiating into each cell constituting a tissue, and refers to a cell having the ability to differentiate into a specific cell under a specific differentiation stimulus. Different from differentiated cells in which cell division stops, stem cells can produce cells identical to themselves through cell division (self-renewal). When a differentiation stimulus is applied, stem cells have differentiation plasticity, and they can differentiate into various cells according to the nature of the stimulus. The stem cells used in the present invention have the characteristics of stem cells, that is, undifferentiated, infinite proliferation, and the ability to differentiate into specific cells, and any cells capable of inducing differentiation into the tissue to be regenerated can be used without limitation.

[0040] Specifically, the stem cells are mesenchymal stem cells.

[0041] As used herein, the term "mesenchymal stem cell" refers to a stem cell having multipotency capable of differentiating into adipocytes, osteocytes, chondrocytes, myocytes, neurons, or cardiomyocytes. Mesenchymal stem cells can be identified by their swirling shape and the expression levels of the basic cell surface markers CD73(+), CD105(+), CD34(-), CD45(-), and also have the functions of regulating immune responses and multipotency.

[0042] For example, the mesenchymal stem cell can be a umbilical cord-derived mesenchymal stem cell (WJ-MSC).

[0043] As used herein, the term "cellular senescence" refers to the expression of the natural aging phenomenon expressed at the cellular level in which the functions of living organs deteriorate over time. For example, it means the arrest or significant delay of growth and division caused by various physical, chemical, and biological stresses (e.g., high oxygen conditions during continuous passage and in vitro culture) received by cells from the inside or outside. In undifferentiated cells such as stem cells, cellular senescence is a concept including a process in which stem cells lose their undifferentiated phenotype and lose their unipotent or pluripotent properties.

[0044] As used herein, the term "inhibition, delay, or reversal of cellular senescence" refers to a series of processes including artificially and temporarily inhibiting cellular senescence, delaying the rate of senescence, or reversibly restoring the biological functions lost by cells due to senescence to have a phenotype similar to that of non-senescent young cells. Therefore, the term "inhibition of cellular senescence" can also be expressed as "delaying cellular senescence" or "inducing the reversal of cellular senescence".

[0045] According to the present invention, when a constant energy shock is applied to cells flowing according to the method of the present invention, the size of the cells decreases, the actin density of the cytoskeleton increases, reactive oxygen species decrease, and the expression of senescence-related proteins or genes (including β-galactosidase and γ-H2AX) is significantly inhibited, so as to effectively inhibit, delay, or reverse the cellular senescence process.

[0046] According to another embodiment of the present invention, the present invention provides a cell in which senescence is inhibited by the above method.

[0047] According to the present invention, cells whose senescence is inhibited by the method of the present invention have high activity and proliferation ability compared with the number of passages, and thus can be effectively used as effective cell therapeutics or research cells even after long-term culture. Specifically, by maintaining the undifferentiated state and maintaining multipotency, stem cells can also increase their value as therapeutic cells for various degenerative diseases caused by irreversible tissue loss.

[0048] According to a specific embodiment of the present invention, the cell can be a cell in which the activity or expression of a protein selected from β-galactosidase, γ-H2AX, and combinations thereof is inhibited.

[0049] As used herein, the term "inhibition of activity or expression" means a decrease in the activity or expression level of a specific protein or the gene encoding it. Thus, inhibition of the activity or expression of a specific protein or gene means that the activity or expression of the protein or gene is reduced to such an extent that detection of the protein or gene is impossible or they are present at non-significant levels and their biological functions are significantly reduced. For example, compared to control cells to which the method of the present invention has not been applied, the activity or expression of the protein or gene in cells in which the activity or expression of an aging-related protein or gene is inhibited by the method of the present invention is reduced by about 10% or more, reduced by about 20% or more, reduced by about 30% or more, reduced by about 40% or more, reduced by about 50% or more, or reduced by about 60% or more, but ranges other than these are not excluded.

[0050] As described above, the cells of the present invention in which the activity or expression of an aging-related protein or gene is significantly inhibited are new cells having a new protein expression profile that did not exist previously.

[0051] According to another embodiment, the present invention provides a method for maintaining or enhancing the pluripotency of stem cells, the method comprising the steps of:

[0052] Flowing the isolated stem cells; and

[0053] Impacting the stem cells onto an impact surface mounted on the flow path of the stem cells to apply a physical impact to the stem cells.

[0054] Since the flow, impact, application of physical impact, and the stem cells to which the physical impact is applied have been described above, their detailed description will be omitted to avoid excessive repetition.

[0055] According to another embodiment, the present invention provides a device for inhibiting, delaying, or reversing the senescence of isolated cells, the device comprising:

[0056] An inlet through which a fluid containing cells is injected;

[0057] A microchannel through which the fluid injected through the inlet flows;

[0058] An impact surface mounted on the flow path of the microchannel; and

[0059] An outlet for discharging the cells that flow along the microchannel and impact onto the impact surface.

[0060] According to an embodiment of the present invention, the device further comprises: a second inlet, through which additional fluid with or without cells can be injected simultaneously or sequentially with the injection of the fluid containing cells. In an embodiment, the additional fluid may be a cell-free buffer solution.

[0061] According to an embodiment, when the device includes both an inlet and a second inlet, each of the inlet and the second inlet may have a separate connection portion connected to the microchannel. The two connection portions may join at a point before reaching the microchannel and are connected to the microchannel through a single flow path.

[0062] The features and advantages of the present invention are summarized as follows:

[0063] (a) The present invention provides a method for delaying the senescence of various cells and maintaining high biological activity compared to the number of passages using a microfluidic platform.

[0064] (b) The present invention further increases the value of stem cells as therapeutic cells for various degenerative diseases by maintaining the undifferentiated state of stem cells and their pluripotency even during long-term culture.

[0065] (c) The present invention can be used as a useful research tool for the senescence mechanism at the cellular level by not only delaying cellular senescence but also reversibly restoring the senescence that has occurred and the loss of biological functions of cells. Description of the Drawings

[0066] Figure 1 is a schematic diagram showing the structure and operation method of a microfluidic device according to an embodiment of the present invention.

[0067] Figure 2 is a schematic diagram showing the polymerization state (left) and depolymerization state (right) of tubulin and the state of the cytoskeleton in a cell, wherein the cytoskeleton is denatured (right) by applying physical shock to senescent stem cells according to the method of the present invention.

[0068] Figure 3 is a schematic diagram showing low-density F-actin in the cell membrane of senescent cells (control) with increased cell size (left) and increased density of F-actin in the cell through reorganization of the cytoskeleton by applying physical shock according to the method of the present invention (right).

[0069] Figure 4 is a photograph showing the cell morphology of each flow condition over time observed by a high-speed microscope when cells flow in the microfluidic device of the present invention under different flow conditions (i.e., different Reynolds numbers Re of 81, 244, or 285).

[0070] Figure 5It is a photograph showing the cell morphology observed by a high-speed microscope near the impact surface in a microfluidic device when umbilical cord-derived mesenchymal stem cells flow under different flow conditions (i.e., different Reynolds numbers (Re) of 81, 244, or 285) in the microfluidic device according to an embodiment of the present invention.

[0071] Figure 6 It is a graph showing the proliferation rate of each cell after 24 hours, 48 hours, and 72 hours, respectively, when umbilical cord-derived mesenchymal stem cells flow under different flow conditions (i.e., Re of 81, 244, or 285) to impact the impact surface in the microfluidic device, compared with the control group (P18) of 18 passages of umbilical cord-derived mesenchymal stem cells not treated with the microfluidic device.

[0072] Figure 7 It is shown after performing Figure 6 the same experiment, a graph and photograph showing the level of intracellular reactive oxygen species (ROS) (one of the senescence markers) stained with DCF-DA for each cell, compared with the control cells (P18).

[0073] Figure 8 It is shown after performing Figure 6 the same experiment, a graph showing the expression changes of the genes OCT4, SOX2, and Klf4, which are undifferentiated markers, for each cell (Re = 81, 244, or 285), compared with the control cells (P9 and / or P18).

[0074] Figure 9 It is shown after performing Figure 6 the same experiment and then measuring the mRNA levels of Oct4, Sox2, and SSEA-4, which are undifferentiated markers, in the cells treated under the flow condition where Re is 244 by immunohistochemical staining, a photograph and graph showing the results compared with the control cells (control).

[0075] Figure 10 It is a graph showing the results of measuring cell cycle changes in the cells treated by the microfluidic device according to the present invention (Re = 244) and the control cells.

[0076] Figure 11 It is a photograph analyzing Figure 10 the cells by immunohistochemical staining of the expression pattern of actin and also measuring the size of the cells over time.

[0077] Figure 12 It is shown by Figure 10 immunohistochemical staining in the cells to analyze the expression patterns of vinculin and tubulin, and the obtained photograph.

[0078] Figure 13 It is a photograph showing the migration of cells over time of umbilical cord-derived mesenchymal stem cells at the 7th passage (P7) and 18th passage (P18) measured by wound healing assay, and umbilical cord-derived mesenchymal stem cells at the 18th passage (P18HP) treated by physical shock (Re = 244) according to the method of the present invention.

[0079] Figure 14 It is a micrograph of umbilical cord-derived mesenchymal stem cells at the 6th passage, 11th passage and 18th passage (P6, P11 and P18) and umbilical cord-derived mesenchymal stem cells at the 18th passage treated by physical shock (Re = 244) according to the method of the present invention through a microfluidic device, and a graph showing the cell area (i.e., the cell size of the cells).

[0080] Figure 15 It is the Figure 14 electron micrograph of cells stained with SA-β-gal to confirm the expression of the senescence marker protein β-galactosidase in the cells and a graph comparing the amount of stained cells measured from the photograph.

[0081] Figure 16 It is a photograph and a graph showing the level of reactive oxygen species (ROS) in the Figure 15 cells by the change amount of DCF-DA (dichlorofluorescein diacetate).

[0082] Figure 17 It is a photograph showing β-galactosidase of senescent stem cells at the 19th passage (P19) measured by SA-β-Gal staining and cells treated by the microfluidic device (Re = 244) according to the present invention.

[0083] Figure 18 It is a graph and a photograph showing SA-β-gal stained cells in each of human neonatal foreskin-derived fibroblasts (BJ) at the 18th passage (BJ P18) and cells treated by the microfluidic device according to the present invention (Re = 244).

[0084] Figure 19 It is a graph and a photograph showing the expression pattern of the senescence marker γ-H2AX in umbilical cord-derived mesenchymal stem cells at the 18th passage (P18) analyzed by histochemical staining and cells obtained by passing the cells through the microfluidic device according to the method of the present invention (Re = 244), and a graph comparing their fluorescence intensities.

[0085] Figure 20 It is the result of analyzing the degree of gene mutation among three groups of control cells (control), cells to which the microfluidic device of the present invention is applied (HP), and electroporated cells (EP) by Venn diagram analysis.

[0086] Figure 21 Shows the results of a comparative analysis of classifying genes in which mutations co-occur in three groups of cells into tumor maps (genes with many mutations) and SNV (single nucleotide variants, genes in which one nucleotide mutates) categories Figure 20 by mutating in which of the three groups of cells co-occur.

[0087] Figure 22 Is a Venn diagram showing the number of upregulated genes that show significantly increased expression compared to the expression of control cells by bioinformatics and downregulated genes that show significantly decreased expression in the cells (HP) to which the microfluidic device of the present invention is applied and Figure 20 among the three groups of cells of the electroporated cells (EP).

[0088] Figure 23 Is a diagram showing the patterns and types of upregulated genes (top two diagrams) with increased expression and downregulated genes (bottom two diagrams) with decreased expression in the cells (HP) that have passed through the microfluidic device of the present invention analyzed by gene ontology (GO), etc.

[0089] Figure 24 Is a diagram showing the results of the expression pattern of genes in the cells (HP) that have passed through the microfluidic device of the present invention by heat map analysis compared to the expression level of genes in control cells.

[0090] Figure 25 Is a photograph showing the wound healing process of the injured tissue over time after treating the injured tissue with each of the stem cells at passage 6 (P6), passage 11 (P11), and passage 18 (P18) that have not been treated with any physical shock according to the invention, treating the cells passing through the microfluidic device of the present invention (Re = 244), and treating only with PBS buffer solution not including any cells, and the control is not treated with any cells or PBS at all.

[0091] Figure 26 Is a diagram showing Figure 25 the relative wound area of the wound healing model over time.

[0092] Figure 27 Is Figure 25 a photograph of H&E staining of the wound healing model for confirming tissue necrosis on the 6th day after the wound.

[0093] Figure 28 Is Figure 25 a photograph of MT (Masson-Trichome) staining of the wound healing model for confirming the tissue-to-tissue binding ability on the 6th day after the wound. Detailed Description of the Invention

[0094] In the following, the present invention will be described in more detail by way of examples.

[0095] These examples are only used to illustrate the present invention in more detail, and it will be apparent to those of ordinary skill in the art that, according to the gist of the present invention, the scope of the present invention is not limited by these examples.

[0096] Examples

[0097] [Experimental methods]

[0098] 1. Culture of umbilical cord-derived mesenchymal stem cells

[0099] Add 1 mL of 0.1% trypsin / EDTA to umbilical cord-derived mesenchymal stem cells (WJ-MSC) at passage 18 cultured in a 100 mm culture dish (SPL, #20100), and then incubate in an incubator at 5% CO2 and 37 °C for 3 minutes. Thereafter, add 9 mL of α-MEM serum-free medium to a 15 mL tube (SPL), and then centrifuge at 1,000 rpm for 3 minutes. After discarding the supernatant therefrom, add a culture solution containing 1% P / S and 10% FBS to the α-MEM in the 100 mm culture dish (SPL), inoculate 1×10 6 cells each, and culture for 48 hours before the experiment.

[0100] 2. Cell microfluidics experiment using a microfluidic device

[0101] Add 1 mL of 0.1% trypsin / EDTA to the cells cultured in a 100 mm culture dish, and incubate in an incubator at 5% CO2 and 37 °C for 3 minutes. Then, add serum-free medium thereto and centrifuge at 1,000 rpm for 3 minutes to remove the supernatant. After adding 1 mL of medium supplemented with FBS to the tube from which the supernatant has been removed, add 1 mL to 3 mL of the medium containing cells to a 10 mL syringe (BD, #REF_302149), and connect the tube to the inlet of an injection pump (Levodix, Fusion 100, Touch). After connecting the microfluidic device thereto, perform the experiment while changing the Reynolds number from Re = 75 to Re = 290. Thereafter, after recovering the sample from the outlet, centrifuge under the same conditions as above, and count the number of cells. Use a microscope (Karl Zeiss, Observer A1) and a high-speed camera (VisionResearch, Phantom V710L) to capture cell deformation at up to 580,000 frames per second. Use ImageJ (NIH) to post-process the captured images.

[0102] 3. Quantitative real-time reverse transcription polymerase chain reaction analysis

[0103] The cell culture medium was removed from the cells cultured in a 100 mm culture dish, washed 2 to 3 times with 1X PBS, 1 mL of 0.1% trypsin / EDTA was added, and incubated in an incubator at 5% CO2 and 37 °C for 3 minutes. Then, serum-free medium was added thereto and centrifuged at 1,000 rpm for 3 minutes to remove the supernatant. 300 μL of Trizol (Invitrogen) was added to the tube from which the supernatant had been removed, and RNA was extracted using the direct-zol RNA Miniprep plus (Zymo, #R2070S) kit. The concentration of the extracted total RNA was measured using a Nanodrop spectrophotometer (IMPEN, NanoPhotometer N60 / N50), the RNA was quantified, and then cDNA was synthesized using 2 μg of total RNA and M-MLV reverse transcriptase. qPCR was analyzed using 2x SYBR Green, and the mRNA expression was calculated using GAPDH as a reference value.

[0104] Quantitative real-time reverse transcription polymerase chain reaction was performed using 2x SYBR green mix (EBT-1802), and the experimental results were normalized based on the expression level of GAPDH. The primers used are shown in Table 1. In addition, a total volume of 20 μL was analyzed by real-time PCR (7500, Amersham Pharmacia Biotech).

[0105] (Table 1)

[0106]

[0107]

[0108] 4. β-galactosidase (SA-β-gal) activity assay

[0109] Preparation of umbilical cord-derived mesenchymal stem cells at senescence (passages 17 to 26) and cells treated with a microfluidic device, and SA-β-galactosidase assay was performed according to the previously reported method (Nature protocols, 2009.4(12):p.1798). Briefly, the cells were cultured in a 35 mm culture dish (SPL) until the proliferation state reached 80%, the medium was removed, 1 mL of 1×PBS (Veratech) was added, and then washed twice at 100 rpm for 5 minutes each time. Then, 1 mL each of 2% paraformaldehyde and 0.2% glutaraldehyde was added, and the resulting product was fixed for 15 minutes. After discarding the fixative, 1 mL of 1×PBS was added and washed twice at 100 rpm for 5 minutes each time. After adding 1 mL of the prepared SA-β-gal staining solution, the resulting product was incubated at 37 °C for 15 hours without CO2. Thereafter, after discarding the SA-β-gal staining solution, 1 mL of 1×PBS was added, washed twice at 100 rpm for 5 minutes each time, 1 mL of 100% MeOH (Samjin Industries) was added, and the resulting product was left at room temperature for 30 minutes. Then, after discarding the 100% MeOH, 1×PBS was added and observed with an optical microscope (Fusion 100, Chemyx). The composition of the SA-β-gal staining solution was as follows: 200 mM citric acid / phosphate, 100 mM K4[Fe(CN)6]·3H2O, 100 mM K3[Fe(CN)6], 5 M NaCl, 1 M MgCl2, X-gal 50 mg / mL. SA-β-gal positive cells were shown as blue.

[0110] 5. Evaluation of Reactive Oxygen Species Generation (H2DCF-DA)

[0111] To measure the accumulation of intracellular reactive oxygen species (ROS), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) reagent was used. After culturing umbilical cord-derived mesenchymal stem cells in α-MEM (1% P / S, 10% FBS) medium at 37 °C, the medium was discarded and the resulting product was washed thoroughly with 1×PBS once or twice. Then, after adding α-MEM (1% P / S, w / o FBS) culture solution, the solution was added such that the final concentration of H2DCFDA was 10 μM, and the cells were incubated at 37 °C under 5% CO2 for 30 minutes. After discarding the medium to which 10 μM H2DCFDA was added, the resulting product was washed with 1×PBS once or twice, and umbilical cord-derived mesenchymal stem cells were directly observed using a fluorescence microscope (Nikon Eclipse TE2000-E).

[0112] 6. Immunocytochemistry

[0113] The cells were fixed with 4% paraformaldehyde, washed 3 times with PBS, and 1× Triton X-100 was added thereto, followed by incubation for 15 minutes. After washing 3 times with 1x PBS, the resulting product was blocked with 10% bovine serum albumin at room temperature for 1 hour and 30 minutes, and then incubated with the primary antibody. Then, it was incubated with the secondary antibody at room temperature for 40 minutes and stained with DAPI. Then, the resulting product was captured using a Zeiss LSM 800 confocal laser scanning microscope.

[0114] 7. Cell cycle analysis using flow cytometry

[0115] The cells were cultured in a 100 mm culture dish (SPL) until the proliferation state reached 80%. The medium was removed from them, and 1 mL of 1× PBS (Veratech) was added thereto, followed by washing twice at 100 rpm for 5 minutes each time. After that, 1 mL of 0.1% trypsin / EDTA was added, and the resulting product was incubated in an incubator at 37 °C and 5% CO2 for 3 minutes. After adding serum-free medium and centrifuging at 1,000 rpm for 3 minutes, the supernatant was removed. After discarding the fixative after the first centrifugation, 1 mL of 1× PBS was added, and the resulting product was washed twice at 100 rpm for 5 minutes each time. Then, 1 mL of 70% EtOH was added, and the cells were fixed at room temperature for 30 minutes. Then, after centrifuging at 1,000 rpm for 3 minutes, the EtOH was removed. Thereafter, 300 μL of PI / RNase staining buffer (5 mg / mL RNase, No. 51-6551AZ) was added, followed by reacting at room temperature for 30 minutes, and the cell cycle was analyzed using a flow cytometer (Beckman Coulter / CytoFLEX).

[0116] 8. Analysis of gene mutations

[0117] The cells at the 9th passage among the umbilical cord-derived mesenchymal stem cells were cultured in a 100 mm culture dish (SPL) until the proliferation state reached 80% to 90%. After removing the culture medium, 1 mL of 1×PBS (Veratech) was added, and the resultant was washed two or three times. Thereafter, 1 mL of 0.1% trypsin / EDTA was added, and the resultant was incubated in an incubator at 5% CO2 and 37 °C for 3 minutes. Thereafter, serum-free medium was added, centrifuged at 1,000 rpm for 3 minutes, and the supernatant was removed. Then, the cells at the 10th passage obtained were electroporated using a NeonTM transfection system (MPK5000, InvitrogenTM) under the following conditions. That is, the cells were stimulated with a voltage of 1650 V and a pulse width of 10 ms. Then, the obtained umbilical cord-derived mesenchymal stem cells were inoculated in a 100 mm culture dish at a number of 6 1×10 cells, cultured in an incubator for 2 to 3 days, and the cells were separated and stored at -20 °C. Thereafter, whole-exome sequencing was performed to selectively analyze only the exon regions within the genes by NovaSeq 6000 analysis (Macrogen) to analyze the gene expression of the control cells, which were the untreated umbilical cord-derived mesenchymal stem cells at the 10th passage, the cells to which the electroporation method was applied, and the cells through the microfluidic device according to the method of the present invention.

[0118] 9. Structure and operation method of the microfluidic device

[0119] Figure 1 is a schematic diagram showing the structure of the microfluidic device according to the embodiment and the process of recovering cells by applying an impact to the cells after they flow through them to cause deformation.

[0120] Refer to Figure 1, The microfluidic device according to the embodiment includes one or more syringes (Syrige 10mL LUER Lock, 302149, BD) for injecting target cells and a fluid such as a buffer solution into the microfluidic device, one or more injection pumps (Chemyx Fusion 100Tuch) for pushing one or more syringes at a constant speed to inject the cells and / or buffer solution in each syringe into the microfluidic device at a constant speed, a Luer adapter (LS25, Luer fitting, 25ga (red) × 0.5in (12mm), tubing (PEEK Tubing, 1 / 32” OD (outer diameter) × 0.020” ID (inner diameter)) for connecting the syringe and a power supply for supplying power to the injection pump, a camera (Optinity auto focus HD camera, KCS-50F) for photographing the movement and shape change of cells in the microfluidic device, etc. Syringes can also be provided for injecting target cells and buffer solution separately, or alternatively, one syringe can be used for injecting the fluid containing target cells into the microfluidic device simultaneously. If necessary, other elements of the microfluidic device can also be provided or excluded, and the diameter and length of the tubing can be appropriately adjusted according to the example.

[0121] The operation steps of the microfluidic device constructed as described above can be divided into the following three steps:

[0122] First, cells and phosphate buffered saline aqueous solution (PBS) are separately placed into two syringes (each about 10 mL in volume), and then each syringe is connected to the pump ( Figure 1 in phase I).

[0123] Second, the pumps connected to each syringe are operated by setting the flow rate to a Reynolds number (Re) of, for example, 1 to 500. Thus, the cells and PBS contained in the syringes can be injected into the microchannels in the microfluidic device and flow along the microchannels at a constant speed ( Figure 1 in phase II).

[0124] Third, the cells moving at a constant speed along the microchannel are impacted into the inner wall (i.e., the impact surface) extending perpendicular to the cell moving direction, so as to receive a physical impact of an appropriate force, and then the deformed cells are recovered through the outlet ( Figure 1 in phase III).

[0125] 10. In vivo analysis of wound healing in a wounded mouse model

[0126] To evaluate the in vivo wound healing effect, 6-week-old BALB / c nude female mice (CAnN.Cg-Foxn1nu / CrljOri SPF / VAF immunodeficient mice) were obtained from Orient Bio Inc. (Seongnam-si, Gyeonggi-do, Korea). The experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (identification number: KU20132). To properly prepare for the experiment, the mice were bred in a room with well-controlled temperature and humidity under 12-hour light and 12-hour dark conditions for 1 week before the experiment. The mice were divided into the following 6 groups:

[0127] (1) Control group, without any treatment;

[0128] (2) 1X PBS (buffer solution) treatment group;

[0129] (3) Treatment group with umbilical cord-derived mesenchymal stem cells at passage 6 (WJ-MSC_P6);

[0130] (4) Treatment group with umbilical cord-derived mesenchymal stem cells at passage 11 (WJ-MSC_P11);

[0131] (5) Treatment group with umbilical cord-derived mesenchymal stem cells at passage 18 (WJ-MSCP18); and

[0132] (6) Treatment group with umbilical cord-derived mesenchymal stem cells at passage 18 (WJ-MSCP18-HP) treated by physical shock with the microfluidic system according to the embodiment. Each group had 4 mice. Before making the wounds, each mouse was anesthetized by intraperitoneal injection of 60 mg / kg dose of Alfaxan (Careside Co., Ltd., Gyeonggi-do, Korea). Specifically, the mice were anesthetized, and 2 wounds were made on the back of each mouse using a sterilized biopsy punch (diameter 8 mm, Kai Industries, Tokyo, Japan). Then, 1X PBS (for the control group) or 100 μL containing 2×10 6Each in a PBS solution of dispersed WJ-MSCP6, P11, P18, and WJ-MSC18-HP cells at a concentration of cells / mL. To prevent contamination, the wound was sealed with silicon (0.5 mm thick), Tegaderm tape (1622W, 3M), and dressing (DUPOL). The size of the wound was determined by a 30-cm scale and recorded by using a digital camera. Six days after injecting the cells or PBS, the mice were sacrificed and the skin tissue around the wound was incised. Then, the incised part was fixed with 4% paraformaldehyde, dehydrated with alcohol, and embedded in paraffin. Then, the tissue was cut perpendicular to the surface of the wound at a thickness of 4 μm, and the cut tissue was placed on a slide pre-coated with 0.1% w / v poly-L-lysine (Sigma, St. Louis, MO). Subsequently, to visualize the lesions and tissue recovery, each section was stained with hematoxylin and eosin, and the degree of surface remodeling was evaluated. In addition, the collagen synthesis rate was estimated by using Masson's trichrome staining method. To obtain tissue images, the slides were scanned by using a digital slide scanner (3D-Histech, Budapest u.3., Hungary).

[0133] [Experimental Results]

[0134] 1. Results of High-Speed Microscopic Observation

[0135] While injecting cells into the microfluidic device according to an embodiment of the present invention and then flowing the cells therein by applying flow rates (i.e., Reynolds numbers Re from 75 to 290) under various flow conditions, changes in the single cells flowing in the microfluidic device relative to the vicinity of the impact surface (so-called, in the deformation region) of the microfluidic device were observed by using an ultra-fast microscopic camera (refer to Figure 4 and Figure 5 ).

[0136] Refer to Figure 4 , when the flow rate was relatively low (Re = 81), the cells immediately moved to the outlet after colliding into the impact surface. However, when the flow rate increased, for example, when Re was 244 or 285, the cells did not immediately move to the outlet but stayed for a while due to the eddy formed around the stagnation point near the impact surface, which maximized the deformation of the cells.

[0137] Refer to Figure 5 , as Re increased, the deformation of the cells near the impact surface also increased. In other words, when Re = 81, the cells maintained an oval shape, but when Re = 244, the cells became flat, and when Re = 285, the cells became even flatter.

[0138] The appropriate Re required for cell deformation has been shown to vary depending on the cell type, but when Re is too high, the cells will lyse.

[0139] 2. Viability of umbilical cord-derived mesenchymal stem cells according to the flow rate (Re) of the microfluidic device

[0140] After flowing the cells in the microfluidic device according to the examples by applying flow rates corresponding to various Re (75 to 290), the change in the proliferation rate of the stem cells according to each Re was measured after 24 hours, 48 hours, and 72 hours, and the results are shown in Figure 6 in.

[0141] As Figure 6 shown in, the control P18 stem cells, i.e., senescent stem cells at passage 18, exhibited a proliferation rate similar to that of the cells flowing at Re = 81. In other words, at Re = 81, almost no impact was applied to the cells, and almost no cell deformation effect was observed. On the contrary, at Re = 244, after 24 hours and 48 hours, the cell proliferation rate decreased by about 20% compared to the control group (P18), but after 72 hours, it was similar to the cell proliferation rate of P18 or at Re = 81. In other words, at Re = 244, the cell proliferation rate was partially reduced due to the initial cell deformation, but it was basically equal to that of the untreated control group (P18) or the cell proliferation rate at Re = 81 with almost no cell deformation effect over time, which can be interpreted as an inhibitory effect on cell senescence due to cell deformation and an adverse effect of the reduction in the cell proliferation rate caused by cell deformation. On the contrary, at Re = 285, from the beginning (24 hours) to 72 hours, the cell proliferation rate decreased by 50% or more compared to all other cells. In other words, when Re is too high, there is a higher risk of cell lysis, etc., compared to the anti-aging effect caused by cell deformation.

[0142] From these results, it can be seen that the flow rate or Re in the microfluidic device affects cell viability.

[0143] 3. Analysis of stem cell characteristics and senescence marker expression of umbilical cord-derived mesenchymal stem cells

[0144] After flowing senescent umbilical cord-derived mesenchymal stem cells (WJ-MSC, P19) through the microfluidic device at flow rates corresponding to various Re, when examining the level of reactive oxygen species (ROS) (one of the senescence markers) in the cells by the DCF-DA staining method, as shown in Figure 7 the amount of reactive oxygen species in the cells under all flow rate conditions was significantly reduced compared to that in the control group.

[0145] In addition, as the flow velocity increases, the amount of reactive oxygen species (ROS) in the cells further decreases. Specifically, at Re = 285, the ROS decreases by about 40% compared to the ROS in the control group.

[0146] In addition, the expressions of the genes OCT4, SOX2, and Klf4, which are stem cell characteristic indices as stem cells, were measured, and the results are shown in Figure 8 in.

[0147] As shown in the left figure of Figure 8 As the flow velocity (Re) increases, compared with the expression in the control group, the expressions of these genes increase. Specifically, at Re = 244 and 285, compared with the expression in the control group (P18), the expressions of the stem cell characteristic marker genes increase by 4 times or more. In addition, as shown in the right figure of Figure 8 As shown in the right figure, compared with the expression of umbilical cord-derived mesenchymal stem cells at the middle passage (P9) as the control group, the expressions of OCT4, SOX2, and Klf4 in the cells treated with the microfluidic device according to the present invention increase by 2 to 4 times.

[0148] In addition, Figure 9 The immunohistochemical (IHC) staining results of Figure 9 also confirmed that compared with the expression level in the control group, the expression levels of Oct4, Sox2, and SSEA-4 at Re = 244 were significantly increased. In addition, as shown in the figure of

[0149] The experimental results show that when physical shock is applied to the cells in the method of the present invention, the stem cells continue to be undifferentiated and thus maintain stem cell characteristics. In other words, cell senescence is inhibited.

[0150] 4. Cell cycle analysis of umbilical cord-derived mesenchymal stem cells

[0151] The cell cycle is divided into the cell division phase and the interphase for preparing cell division. In addition, the interphase, which occupies about 90% of the cell cycle, is further divided into the G1 phase, S phase, and G2 phase. Generally, cells survive in sequence through the life of G1, S, G2, and M (mitosis), but specifically, depending on the cells, there are various interphase durations. In the present invention, in order to examine whether the physical shock applied to the cells participates in the regulation of the cell cycle, the cells were flowed at a flow velocity of Re = 244, and analyzed relative to the cell cycle by using the fluorescence-activated cell sorting (FACS) method, and the results are shown in Figure 10 in.

[0152] As shown in Figure 10As shown, in the cell cycles of both the control group and the experimental group, since no arrest in the G2 phase, M phase, or S phase was observed, the physical shock method using the microfluidic device according to the present invention does not alter the cell cycle.

[0153] 5. Expression of senescence markers in umbilical cord-derived mesenchymal stem cells and human fibroblasts

[0154] When metabolic changes occur due to cellular senescence, cells exhibit typical external characteristics such as an increase in size and volume. Therefore, after passing umbilical cord-derived mesenchymal stem cells at passage 18 through the microfluidic device of the present invention, for size, the cells (Re = 244) were compared with umbilical cord-derived mesenchymal stem cells at passage 6 (P6), passage 11 (P11), and passage 18 (P18) that did not pass through the microfluidic device.

[0155] As shown from Figure 14 the microscope photographs, compared with the cells (P18) in the same channel that did not pass through the microfluidic device of the present invention, the cells passing through the microfluidic device (Re = 244) exhibited a size 4 times or smaller. Additionally, referring to the bottom graph in Figure 14 showing the size comparison of the cells, the cells (Re = 244) passing through the microfluidic device of the present invention had a size smaller than that of the cells at intermediate passage (P11) but slightly larger than that of the young cells (P6). Thus, the senescent cells at passage 18 passed through the microfluidic device of the present invention and exhibited a significantly reduced size down to that of the young cells.

[0156] On the other hand, by comparing and experimenting with cells using the X-gal and DCF-DA staining methods to examine the levels of β-galactosidase, one of the cell senescence markers, and intracellular ROS, the results showed in Figure 15 and Figure 16 in.

[0157] Referring to Figure 15 the number of positive cells for Sa-β-Gal among the cells (Re = 244) passing through the microfluidic device of the present invention decreased to 1 / 3 or less compared to the number in the control group (P18), but was slightly higher than the number of the younger cells of P6 or P11.

[0158] Additionally, regarding DCF-DA expression, referring to Figure 16 the cells (Re = 244) passing through the microfluidic device of the present invention exhibited a reduction in the content of ROS of approximately 90% or more compared to the content of ROS in the control group (P18).

[0159] In addition, under the same experiment and confirmed by SA-β-Gal staining, the β-galactosidase expressed by senescent umbilical cord-derived mesenchymal stem cells (WJ-MSC, P19) at passage 19 and the cells (Re = 244) passing through the microfluidic device of the present invention are the same as Figure 15 and Figure 16 , and the results are similar (refer to Figure 17 ).

[0160] In addition, referring to Figure 18 , after using human neonatal foreskin-derived fibroblasts (BJ P18) at passage 18 as a control group and applying an impact to the cells under the same conditions (Re = 244) as above, when measuring the β-galactosidase positive cells in the two cell groups, the number of Sa-b-Gal positive cells in the cells (Re = 244) to which the impact was applied also decreased by nearly 20%.

[0161] 6. γ-H2AX expression of umbilical cord-derived mesenchymal stem cells using a microfluidic device

[0162] When double-strand breaks occur in eukaryotic DNA, one of the initial reactions that occur in cells is the phosphorylation of H2AX (one of the H2A molecules). In other words, γ-H2AX is one of the main markers for cell senescence. Therefore, the expression of γ-H2AX in the cells (Re = 244) passing through the microfluidic device of the present invention was measured. As shown in Figure 19 , its expression rate decreased by 50% or more compared to the expression rate of the control group (P18).

[0163] 7. Actin expression in umbilical cord-derived mesenchymal stem cells using a microfluidic device

[0164] It has been reported that the reorganization of the cytoskeleton regulates various activities such as cell survival, proliferation, death, cell motility, and protein selection (FEBS Letters 582 (2008) 2120-2127). To investigate whether the reorganization of the cytoskeleton by the method of the present invention is related to the reduction of phenotypes related to cell senescence, umbilical cord-derived mesenchymal stem cells were passed through the microfluidic device at Re = 244, and the expression of actin (one of the cytoskeletons) in the recovered cells was compared by immunohistochemical staining. As a result, referring to Figure 11 , compared with the cells of the control group that did not pass through the microfluidic device, the cells (Re = 244) passing through the microfluidic device of the present invention showed different actin expression patterns and cell sizes over time.

[0165] In addition, Figure 12It is a photograph analyzing the expression patterns of actinin and tubulin in cells by immunohistochemical staining. Tubulin is also a protein that constitutes microtubules, which are cytoskeletal fibers involved in the formation of the cytoskeleton. Refer to Figure 12 Compared with the cells of the control group, the cells passing through the microfluidic device of the present invention (Re = 244) showed clearer expression of actinin and tubulin.

[0166] Without intending to be bound by a specific theory, the above results support that when a certain physical impact is applied to cells according to the method of the present invention, temporary changes occur in the cytoskeletal structure of the cells. Therefore, through the inevitable subsequent reorganization of the cytoskeleton, cell senescence is inhibited, delayed, or reversibly reversed.

[0167] 8. Analysis of gene mutations

[0168] Figure 20 It is a Venn diagram showing that when analyzing gene mutations by subjecting the 10th passage cells of umbilical cord-derived mesenchymal stem cells to the microfluidic device of the present invention or by subjecting the 10th passage cells to electroporation and analyzing the comparison with the cells of the untreated control group, 94.9% of the mutations between the control group and the group treated with the microfluidic device of the present invention, and 95.3% of the mutations between the control group and the electroporation group are the same. Therefore, more than 95% of the gene mutations among all groups are shared. Additionally, as a result of comparative analysis to check for common mutations among the three groups, as Figure 21 shown, mutations of the same genes in the two groups were confirmed. This result confirms that the method of applying physical impact according to the present invention to cause actin depolymerization in cells and thus cause reorganization of the cytoskeleton does not cause significant changes in gene expression.

[0169] 9. Bioinformatics analysis

[0170] By using the microfluidic device according to the present invention for bioinformatics analysis, the differences in gene expression patterns were analyzed in cells that were respectively subjected to electroporation and physical impact on the 7th passage cells of umbilical cord-derived mesenchymal stem cells, as well as in the cells of the untreated control group.

[0171] Specifically, RNA was extracted from three types of cells respectively, and then the RNA was used to analyze total mRNA in the NextSeq500 / 550 method. Subsequently, to compare the expression differences of the analyzed RNA, genes with significant differences in expression levels were selected from the control group and the experimental group by z-score and p-value respectively, and the results are shown in Figure 22 and Figure 23 as follows.

[0172] Figure 22It is a graph showing the number of each gene in a Venn diagram after showing upregulated genes with significantly increased expression and downregulated genes with significantly decreased expression in electroporated cells (EP) and cells (HP) to which the microfluidic device of the present invention is applied through bioinformatics analysis.

[0173] Referring to Figure 22 the Venn diagram, the total number of genes (HP-UP) showing increased expression in cells (HP) subjected to physical shock according to the present invention is 198, and 174 of these genes (EP-UP) show increased expression in cells (EP) subjected to electroporation. Therefore, these 174 genes show increased expression in both types of cells. Additionally, the total number of genes (HP-DOWN) showing significantly decreased expression in cells (HP) subjected to the physical shock of the method according to the present invention is 236, and 149 of these genes are also co-expressed in cells (EP) subjected to electroporation.

[0174] Figure 23 It is a graph (right) showing the patterns of genes with increased expression (HP-UP) and genes with decreased expression (HP-DOWN) in cells through the microfluidic device by the method of the present invention by using databases of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology Biological Process (GO_BP), and a perspective view showing the positions of genes in cells by using GO_BP.

[0175] As Figure 23 shown in the two graphs (based on KEGG and GO_BP) at the upper right of Figure 22 most of the genes showing increased expression in Figure 23 are genes highly related to the cell cycle or DNA repair, as schematically shown at the upper left of Figure 23 genes expressing proteins mainly present in the cell, cell membrane, etc. Conversely, Figure 22 the two graphs at the lower right of Figure 23Schematically shown at the lower left, these genes are genes that express proteins mainly located in the matrix outside the cell (i.e., in the extracellular matrix (ECM)), and referring to existing research, genes with reduced expression are mainly related to cellular senescence (please refer to Stefanie Sudhop et al., Age related changes in cell stiffness of tendon stem / progenitor cells and a rejuvenating effect of ROCK-inhibition, Biochemical and Biophysical Research Communications 509 2019 839-844; Pinar Zorlutuna et al., Effect of cellular and ECM aging on human iPSC-derived cardiomyocyte performance, maturity and senescence, Biomaterials 268 2021 120554; Toshie Tsuchiya et al., FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-β2, Biochemical and Biophysical Research Communications 359 2007 108-114; Ting-Hein Lee et al., MFG-E8 mediates arterial aging by promoting the proinflammatory phenotype of vascular smooth muscle cells, Journal of Biomedial Science 2019 26:61; Douglas E. Vaughan et al., PAI-1–regulated extracellular proteolysis governs senescence and survival in Klotho mice, PNAS 7090-7095, May 13, 2014, vol.111, no.19; Michelle R. Dawson et al.,Senescent mesenchymal stem cells remodel extracellular matrix driving breast cancer cells to a more-invasive phenotype, Journal of Cell Science 2020 133, jcs232470.doi:10.1242 / jcs.232470; Rui-Ming Liu et al., Serpine1 induces alveolar type II cell senescence through activating p53-p21-Rb pathway in fibrotic lung disease, Aging Cell 2017 16, pp.1114-1124; Irit Sagietal., The ECM path of senescence in aging: components and modifiers, The FEBS Journal 287 2020 2636-2646; Lester F. Lau et al., The Matricellular Protein CCN1 / CYR61 Induces Fibroblast Senescence and Restricts Fibrosis in Cutaneous Wound Healing, Nature Cell Biology, Author manuscript, available in PMC 2011 January 01; Marco Demaria et al., Unmasking Transcriptional Heterogeneity in Senescent Cells, Current Biology 27, 2652-2660, September 11, 2017 etc.).

[0176] Referring to the above results, physical shock can be applied to cells by the method according to the present invention to inhibit, delay or reverse cell senescence.

[0177] 10. Heatmap analysis of genes

[0178] As described above, by using heat map analysis, genes with different expressions in cells subjected to physical shock through the microfluidic device of the present invention were examined in more detail for DNA repair systems, cell cycles, p53 mechanisms related to cell senescence, etc., and the results showed that in Figure 24 medium.

[0179] Refer to Figure 24 , in cells subjected to the physical shock according to the present invention, most of the genes have increased expressions. Although some of the genes show increased expressions in the direction of senescence, most of them show increased expressions in the direction of reverse senescence (anti-aging), increased or decreased expressions in the direction of cell cycle activation, and decreased expressions of senescence-inducing genes such as TP53.

[0180] As Figure 24 shown in the figure at the top of Figure 24 , BER (base excision repair), MR (mismatch repair), NER (nucleotide excision repair), DSBR (double-strand break repair), etc. are processes that recognize and correct DNA damage and then repair them, and these damages are caused by various stimuli (reactive oxygen species, ultraviolet (UV), X-rays, etc.) and replication errors in the DNA repair system. When this DNA repair process slows down or is poorly performed, cell senescence occurs, and phenomena such as tumor formation and apoptosis occur. All the genes marked in each corresponding region of Figure 24 are genes that express proteins responsible for the functions of each region as described above. In addition,

[0181] Refer to Figure 24 the results, most of the genes (PCNA, POLD1, etc.) involved in regulating the above phenomena show increased expressions in cells passing through the microfluidic device of the present invention. Referring to this result, it is proved that cell senescence is inhibited and delayed by the method of the present invention.

[0182] 11. In vitro wound healing effect

[0183] Figure 13It is a photograph showing the migration over time of cells of umbilical cord-derived mesenchymal stem cells at passage 7 (P7) and passage 18 (P18) assayed by wound healing, and cells of umbilical cord-derived mesenchymal stem cells at passage 18 (P18 HP) treated by physical shock (Re = 244) according to the method of the present invention. Specifically, the above cells were filled in a culture dish at a specific density while keeping a part unoccupied. Then, the dish was cultured under cell culture conditions for a period of time, and the changes in the unoccupied area were observed. As a result, if the unoccupied area was filled with cells due to proliferation, a wound healing effect was presented. That is, the in vitro wound healing effect is one of the functional models for evaluating the migration of cells caused by proliferation. Therefore, if the cells are senescent, the wound healing effect decreases with the reduction of cell migration or proliferation. Refer to Figure 13 , cells (P18 HP) were treated by physical shock (Re = 244) according to the method of the present invention. However, the senescent cells at passage 18 showed similar or better effects compared with the younger cells at passage 7 (P7). On the contrary, compared with the other two types of cells, the cells at passage 18 (P18) not treated by physical shock according to the method of the present invention showed a reduced wound healing effect (i.e., reduced migration) during the same period. That is, it can be assumed that applying physical shock to cells according to the method of the present invention can inhibit, delay or reverse cell senescence.

[0184] 12. Wound healing effect in animal models

[0185] The wound healing effect of senescent mesenchymal stem cells (P18HP) by the microfluidic device (Re = 244) of the present invention was confirmed through an animal model.

[0186] Figure 25 It is a photograph showing the wound healing process over time of the groups treated with mesenchymal stem cells at passage 6 (P6), mesenchymal stem cells at passage 11 (P11), and mesenchymal stem cells at passage 18 (P18) that were not passed through the microfluidic device of the invention, mesenchymal stem cells at passage 18 (P18HP) passed through the microfluidic device (Re = 244) of the present invention, the control group not treated with any cells or PBS, and the group treated only with PBS.

[0187] Refer to Figure 25, senescent mesenchymal stem cells (P18HP) passing through the microfluidic device (Re = 244) of the present invention exhibited wound healing activity similar to that of young stem cells (stem cells at passage 6, i.e., P6). Cells passing through the microfluidic device of the present invention, which are senescent mesenchymal stem cells at passage 18 that have undergone physical shock through the microfluidic device of the present invention, showed a wound healing effect superior to that of young mesenchymal stem cells at passage 11. On the other hand, compared with cells that have undergone the physical shock according to the present invention, senescent mesenchymal stem cells at passage 18 (P18) that have undergone physical shock through the microfluidic device of the present invention still showed a wide wound site and very low wound healing activity even on the 9th day after the wound.

[0188] Figure 26 is a graph showing Figure 25 the relative wound area of the tissue over time. Referring to Figure 26 , on the 3rd day after the wound, the wound area of the tissue treated with cells passing through the microfluidic device according to the present invention was similar to the wound area of the tissue treated with P6 or P18 cells. Therefore, in all these tissues, the wound area hardly decreased. However, starting from the 6th day after the wound, the wound area of all tissues decreased significantly. Among them, when compared with the control group or the group treated with P11 or P18 cells, the tissue treated with cells passing through the microfluidic device of the present invention showed a wound area slightly larger than that of the tissue treated with P6, but a wound area 25% or smaller than that of other groups, and thus had excellent wound healing activity. In addition, from the 9th day after injury, all tissues showed a significantly reduced wound area. Among them, the tissue treated with cells passing through the microfluidic device of the present invention and the tissue treated with P6 cells showed a wound area significantly reduced to about 1 / 3 or more of the wound area of other tissues. In other words, even very senescent cells at passage 18, cells that have undergone physical shock through the microfluidic device of the present invention are reversely senescent and proved to have excellent wound healing activity similar to that of young cells at passage 6. In other words, cells passing through the microfluidic device of the present invention are proved to be reversely senescent.

[0189] As can be seen from Figure 26 shown, since the wound healing activity of all cells was activated from the 6th day after the wound, H&E staining was performed to check whether the intracellular tissue was necrotic due to inflammation of each cell, and the results are shown in Figure 27 . As can be seen from Figure 27 shown, on the 6th day after the wound, the necrosis of the tissue treated with young P6 cells and the cells according to the present invention was relatively very low compared to the necrosis of the tissue of other cells.

[0190] In addition, MT (Masson-Trichome) staining was performed to examine the tissue-to-tissue binding ability with collagen and the like on the 6th day after the trauma, and the results are shown in Figure 28 as follows. As shown in Figure 28 , the tissue treated with the cells according to the present invention and P6 cells has a narrow gap between the tissue and collagen, indicating excellent binding ability. In contrast, the tissue treated with P11 and P18 cells shows a wide gap between the tissue and collagen. Specifically, the tissue treated with P18 cells has a very wide gap. On the contrary, the tissue treated with the cells treated by the physical shock of the microfluidic device according to the present invention shows a narrow gap unlike the gap of P6, but has a much narrower gap between the tissue and collagen than P11, and thus shows improved binding ability.

[0191] Through the above experiments, by applying physical shock through the microfluidic device according to the present invention, cell senescence can be inhibited, delayed or reversed. Therefore, the cells whose senescence is inhibited, delayed or reversed can maintain the same or similar activity as younger cells.

[0192] As described in detail above for the specific parts of the present invention, for those of ordinary skill in the art, this specific description is only a desired embodiment, and it is clear that the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Sequence Listing <110> Konkuk University Industry-Academic Cooperation Foundation Korea University Industry-Academic Cooperation Foundation <120> Method, Device and Cell for Inhibiting, Delaying or Reversing Cell Senescence <130> <150> KR 10-2020-0094616 <151> 2020-07-29 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic_Oct4 F primer <400> 1 cctgaagcag aagaggatca cc 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composition_Oct4 R primer <400> 2 aaagcggcag atggtcgttt gg 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composition_Sox2 F primer <400> 3 gctacagcat gatgcaggac ca 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composition_Sox2 R primer <400> 4 tctgcgagct ggtcatggag tt 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composition_Klf4 F primer <400> 5 catctcaagg cacacctgcg aa 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composite_Klf4 R primer <400> 6 tcggtcgcat ttttggcact gg 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composite_GAPDH F primer <400> 7 gtctcctctg acttcaacag cg 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Composite_GAPDH R primer <400> 8 accaccctgt tgctgtagcc aa 22

Claims

1. A method for inhibiting, delaying or reversing cellular senescence, the method comprising the following steps: Flowing the isolated cells; And Impacting the cells into an impact surface mounted on the flow path of the cells to apply a physical impact to the cells, thereby causing reorganization of the cytoskeleton in the cells without causing cell death, Wherein the step of flowing the isolated cells is carried out by injecting a fluid containing the cells into a microchannel to flow according to predetermined parameters, Wherein the parameter Re is determined by Equation 1: Re = ρVD / μ Equation 1 Wherein, in Equation 1, ρ is the density of the fluid, V is the velocity of the fluid, D is the characteristic length of the fluid, and μ is the viscosity coefficient of the fluid, and Wherein the parameter Re has a value of 75 to 290, and Wherein the isolated cells are stem cells.

2. The method according to claim 1, wherein, The step of flowing the cells is carried out under the condition that the parameter Re has a value of 81 to 285.

3. The method according to claim 1, wherein The step of flowing the cells is carried out under the condition that the parameter Re has a value of 100 to 280.

4. The method according to claim 1, wherein, The distance from the point where the cells start to flow in the microchannel to the impact surface is 0.1 mm to 50 mm.

5. The method according to claim 1, wherein The distance from the point where the cells start to flow in the microchannel to the impact surface is 5 mm to 30 mm.

6. The method according to claim 1, wherein, The angle between the axis of the flow path and the impact surface is 10 degrees to 170 degrees.

7. The method according to claim 1, wherein, The angle between the axis of the flow path and the impact surface is 20 degrees to 160 degrees.

8. The method according to claim 1, wherein The stem cells include mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells or a combination thereof.

9. The method according to claim 8, wherein The stem cells include mesenchymal stem cells.

10. The method according to claim 9, wherein, The mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells WJ-MSC.

11. A method for maintaining or enhancing the pluripotency of stem cells, the method comprising the following steps: Flowing the isolated stem cells; And Impacting the stem cells into an impact surface mounted on the flow path of the stem cells to apply a physical impact to the stem cells, thereby causing reorganization of the cytoskeleton in the stem cells without causing cell death, Wherein the step of flowing the isolated cells is carried out by injecting a fluid containing the stem cells into a microchannel to flow according to predetermined parameters, Wherein the parameter Re is determined by Equation 1: Re = ρVD / μ Equation 1 Wherein, in Equation 1, ρ is the density of the fluid, V is the velocity of the fluid, D is the characteristic length of the fluid, and μ is the viscosity coefficient of the fluid, and Wherein the parameter Re has a value of 75 to 290.

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