Method for producing cytokine-enhanced mesenchymal stem cells

Abstract

To provide a method for increasing an anti-inflammatory action of mesenchymal stem cells without depending on gene modification.SOLUTION: The present invention provides a method for producing mesenchymal stem cells in which expression of a gene encoding a cytokine is enhanced, the method including a stimulation step of irradiating mesenchymal stem cells with a shock wave.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing cytokine-enhanced mesenchymal stem cells, cells produced by the production method, a cell preparation containing the cells, and a method for enhancing cytokines in mesenchymal stem cells. [Background technology]

[0002] Mesenchymal stem cells were discovered as somatic stem cells present in bone marrow and have the ability to differentiate into one or more types of mesenchymal cells, such as bone, cartilage, and fat. Mesenchymal stem cells are also known to exist in fetal appendages such as the placenta, umbilical cord, and fetal membrane, as well as in adipose tissue. Due to their useful properties, including proliferation, differentiation, immunosuppression, and homing, they have attracted attention as a promising cell source for cell therapy.

[0003] With the increase in available somatic stem cells, the technical barriers to regenerative medicine have been lowering in recent years. Cell transplantation, which involves transplanting living cells into patients for treatment, was initially used mainly in bone marrow transplants, where hematopoietic stem cells are transplanted into patients with intractable blood diseases, but its applications are expanding.

[0004] For example, bone marrow mesenchymal cell preparations for patients with acute graft-versus-host disease (GVHD) and spinal cord injury are sold in Japan as regenerative medicine products. Furthermore, cancer immunotherapy is widely used to treat serious illnesses. This is a treatment method in which immune cells are collected from the patient's blood, selected and cultured ex vivo based on their aggressiveness against cancer, and then transplanted back into the patient's body to strengthen their immunity against cancer.

[0005] In addition to treating such serious diseases, somatic stem cell transplantation has recently begun to be used in the treatment of even milder diseases and conditions, particularly in the field of orthopedics, etc. Specifically, for example, in the case of arthropathy caused by injury or aging, mesenchymal stem cells are injected into the joint to relieve pain or restore joint function, and to improve scars, wrinkles, and sagging skin by injecting stem cells and / or adipocytes differentiated and proliferated from stem cells (Patent Document 1).

[0006] In addition, extracorporeal shock wave therapy has been adopted as a new treatment method in the field of orthopedics. Extracorporeal shock wave therapy was originally used to destroy kidney stones and other internal organs by irradiating them from outside the body. Currently, the scope of its application is expanding as an in vivo treatment technique from outside the body, taking advantage of the fact that shock wave irradiation promotes tissue repair. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4749331 Summary of the Invention [Problem to be solved by the invention]

[0008] Mesenchymal stem cells are extremely useful in cell transplantation, and because of their immunosuppressive properties, they are known to be effective in the treatment of inflammatory diseases. However, while mesenchymal stem cells are effective in patients with relatively mild inflammation or mild symptoms, there are cases where administration of mesenchymal stem cells is ineffective in patients with severe inflammation or severe symptoms.

[0009] Cell therapy using genetically modified cells is one method for treating diseases accompanied by severe inflammation or for patients with severe symptoms. Using genetic modification technology, it is possible to artificially regulate the expression of immune-related genes or inflammation-related genes, making it possible to prepare mesenchymal stem cells that can treat severe inflammatory symptoms. However, the production of genetically modified cells requires advanced processing techniques and specialized equipment, which increases the cost of treatment and poses medical economic challenges.

[0010] Therefore, an object of the present invention is to provide a method for enhancing the anti-inflammatory effect of mesenchymal stem cells without relying on genetic modification. [Means for solving the problem]

[0011] As a result of intensive research conducted by the present inventors to solve the above problems, they focused on extracorporeal shock waves, which are originally used to irradiate the inside of a living body from outside the body, and found that when mesenchymal stem cells are irradiated with shock waves at a specific intensity using this extracorporeal shock wave irradiation device, the expression level of genes encoding cytokines is enhanced. The present invention is based on this novel finding and provides the following. [1] A method for producing mesenchymal stem cells in vitro in which expression of genes encoding cytokines is enhanced, the method comprising a stimulating step of irradiating the mesenchymal stem cells with shock waves. [1-1] A method for producing mesenchymal stem cells in which expression of a gene encoding a cytokine is enhanced, the method comprising a stimulating step of irradiating the mesenchymal stem cells with shock waves. [2] The method according to [1] or [1-1], further comprising a culture step of culturing mesenchymal stem cells. [3] The method according to [2], wherein the culture is an adherent culture. [4] The method according to any one of [1] to [3], wherein the frequency of the shock wave irradiation is 50 Hz or less. [5] The method according to any one of [1] to [4], wherein the number of times the shock waves are irradiated is in the range of 5 to 1000 times. [5-1] The method according to any one of [1] to [4], wherein the number of times the shock waves are irradiated is in the range of 5 to 3000 times. [6] The method according to any one of [1] to [5-1], wherein the stimulation applied in the stimulation step is a stimulation of 0.005 mJ to 0.2 mJ per cell in total. [7] The method according to [6], wherein the frequency of the shock waves is 2 Hz to 10 Hz, the number of times the shock waves are applied is 100 to 600, and the total stimulation is 0.015 mJ to 0.17 mJ per cell. [8] The method according to [7], wherein the shock wave is applied 400 to 600 times and the total stimulation is 0.05 mJ to 0.1 mJ per cell. [9] The method according to any one of [1] to [8], wherein the cytokine comprises a TGF-β family protein and / or an interleukin.

[10] The method according to any one of [1] to [9], further comprising a recovery step of recovering mesenchymal stem cells after the stimulation step.

[11] Cells produced by the method according to any one of [1] to

[10] .

[12] A cell preparation comprising the cells described in

[11] .

[13] An anti-inflammatory agent comprising the cells described in

[11] .

[14] A method for enhancing the expression of a gene encoding a cytokine in a mesenchymal stem cell ex vivo, the method comprising a stimulating step of irradiating the mesenchymal stem cell with shock waves.

[15] A cell preparation that enhances the expression of genes encoding cytokines in mesenchymal stem cells in vivo, wherein the administration of the cell preparation is combined with a stimulating step of irradiating the living body with shock waves. [Effects of the Invention]

[0012] According to the production method of the present invention, mesenchymal stem cells in which the expression of genes encoding cytokines is enhanced can be produced.

[0013] According to the cell preparation of the present invention, a cell preparation containing mesenchymal stem cells in which the expression of a gene encoding a cytokine is enhanced can be used for treatment.

[0014] According to the enhancement method of the present invention, it is possible to enhance the expression of genes encoding cytokines in mesenchymal stem cells. [Brief explanation of the drawings]

[0015] [Figure 1] These figures show the results of IL1RN gene expression in mesenchymal stem cells subjected to shock wave stimulation under each condition in Example 1. Figure 1A shows the 2-ΔCt values ​​normalized by the expression level of the GAPDH gene (internal standard). Figure 1B shows the 2-ΔΔCt values ​​normalized by the expression level of the GAPDH gene (internal standard) as well as the results under condition A (non-shock wave irradiation). [Figure 2] These figures show the results of TGF-β1 gene expression in mesenchymal stem cells subjected to shock wave stimulation under each condition in Example 1. Figure 2A shows the 2-ΔCt values ​​normalized by the expression level of the GAPDH gene (internal standard). Figure 2B shows the 2-ΔΔCt values ​​normalized by the expression level of the GAPDH gene (internal standard) as well as the results under condition A (non-shock wave irradiation). DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Method for producing cytokine-enhanced mesenchymal stem cells and method for enhancing cytokines Overview The first aspect of the present invention is a method for producing mesenchymal stem cells in which expression of a gene encoding a cytokine is enhanced, and a method for enhancing expression of a gene encoding a cytokine in mesenchymal stem cells. The method of the present invention includes a stimulation step as an essential step. According to the method of this aspect, the cells described in the second aspect can be prepared.

[0017] 1-2.Definition Terms used in this specification are defined below. As used herein, "mesenchymal cells" refer to cells that constitute mesodermal tissue. Mesenchymal cells include all cells that constitute mesodermal tissue, including, but not limited to, osteoblasts, adipocytes, muscle cells, chondrocytes, and the like. As used herein, mesenchymal cells also include mesenchymal stromal cells and mesenchymal stem cells.

[0018] As used herein, the term "mesenchymal stem cell (MSC)" refers to a cell that meets the following definitions (i) and (ii): (i) They exhibit adhesion to plastic under culture conditions in standard medium (standard medium is a basal medium (e.g., αMEM medium) supplemented with serum, serum replacement reagents, or growth factors). (ii) The surface antigens CD73 and CD90 are positive, and CD45 and CD326 are negative.

[0019] Mesenchymal stem cells are multipotent somatic stem cells that can differentiate into one or more types of cells belonging to mesodermal tissue. As used herein, mesenchymal stem cells include both mesenchymal stem cells obtained from any tissue and mesenchymal stem cells prepared in vitro.

[0020] "Cytokines" are proteins secreted by cells that regulate the function of the immune system. The main function of cytokines is to strengthen lymphocytes such as T cells and NK cells.

[0021] A "shock wave" is a type of sound wave with a wavefront where density and pressure increase rapidly, and refers to a compression wave that propagates through a compressible medium. Generally, the wavefront of a shock wave forms a discontinuous surface. In particular, the shock wave in this specification refers to a shock wave in a liquid medium (underwater shock wave). Generally, the propagation speed of a shock wave is supersonic, but the shock wave in this specification also includes waves traveling at speeds slower than the speed of sound, as long as they have discontinuities in pressure, etc., and are compression waves that are generally included in underwater shock waves.

[0022] As used herein, "significant" refers to statistical significance. Statistically significant refers to a significant difference between the measured value of a test subject and the control value when the difference between the two is statistically processed. For example, the risk rate (significance level) of the obtained value is small, specifically, less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). The "p (value)" shown here indicates the probability that a test statistic will take that value by chance in a distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic will take that value, meaning that the null hypothesis is more likely to be rejected. The statistical processing test method may be any known test method capable of determining the presence or absence of significance, and is not particularly limited. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank sum test, analysis of variance, Tukey's post-hoc test, etc. can be used, but are not particularly limited.

[0023] 1-3.Process The production method of the present invention includes a stimulation step as an essential step, and includes an acquisition step, a culture step, and a recovery step as optional steps. These will be specifically described below.

[0024] The stimulation step herein may be carried out in vitro or in vivo. In this case, "in vitro" here includes any condition carried out outside the body of an individual organism, including, for example, both ex vivo and in vitro. "In vivo" includes any condition carried out inside the body of an individual organism, including, for example, irradiation of shock waves from outside the body to a site in the body into which cells have been administered, as described below. In this specification, the term "irradiation to a living organism" means "in vivo."

[0025] 1-3-1. Acquisition process The obtaining step is an optional step in the method of the present invention, which is a step of obtaining mesenchymal stem cells.

[0026] The method for obtaining mesenchymal stem cells used in this step is not particularly limited and can be appropriately selected depending on the type and purpose of the mesenchymal stem cells used.

[0027] For example, cells isolated from individuals or tissues, commercially available mesenchymal stem cells, cells induced to differentiate from other pluripotent stem cells such as iPS cells or ES cells, or cryopreserved cells thereof can be used.

[0028] The origin of the mesenchymal stem cells used is not particularly limited. The mesenchymal stem cells may be derived from any organism, and mesenchymal stem cells derived from multiple types of organisms or multiple individual organisms may be used in combination.

[0029] Examples of living organisms in this specification include mammals and birds, such as primates including humans and chimpanzees, pet animals such as dogs, cats, and parakeets, livestock animals such as cows, horses, sheep, goats, and chickens, rodents such as mice and rats, and animals kept in zoos. For example, primates such as humans and other mammals can be preferably used.

[0030] The tissue from which mesenchymal stem cells are derived is not particularly limited. For example, since mesenchymal stem cells exist in a variety of tissues, cells derived from any tissue may be used, or a combination of cells derived from multiple types of tissue may be used. Examples of tissues from which mesenchymal stem cells can be derived include epithelial tissue, connective tissue, muscle tissue, nerve tissue, or a combination thereof.

[0031] Specifically, cells derived from fetal appendages such as the umbilical cord and placenta, bone marrow, adipose tissue, synovium, synovial fluid, dental pulp, heart, etc. can be used. More specifically, examples include skin fibroblasts, osteoblasts, tendon and ligament fibroblasts, adipocytes, chondrocytes, tenocytes, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, mucin-producing cells, and cells derived from endocrine glands (e.g., insulin-producing cells such as β islet cells). Furthermore, in the case of blood, which is a type of connective tissue, blood cells, unlike other mesenchymal cells, are cells differentiated from hematopoietic stem cells. However, in this specification, these cells are also considered to be mesenchymal cells, and stem cells derived from these cells are also included in mesenchymal stem cells. Specifically, mesenchymal cells include, for example, dendritic cells, monocytes, natural killer (NK) cells, T cells (e.g., alpha-beta (αβ) T cells, gamma-delta (γδ) T cells, cytotoxic T cells (cytotoxic T lymphocytes: CTL), helper T cells, etc.), B cells, macrophages, neutrophils, eosinophils, etc. Specifically, mesenchymal stem cells derived from fetal appendages, amniotic membrane, adipose tissue, or a combination thereof can be preferably used.

[0032] When the mesenchymal stem cells used are cryopreserved, the storage period is not particularly limited, and can be, for example, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 1 week or more, 10 days or more, 2 weeks or more, 15 days or more, 20 days or more, 3 weeks or more, 30 days or more, 1 month or more, or 1 year or more.

[0033] When the mesenchymal stem cells used are cryopreserved, the method for thawing the cells is not particularly limited. For example, thawing may be performed by slowly increasing the temperature or by rapidly increasing the temperature. Preferably, thawing is performed by rapidly increasing the temperature. In this case, the method is not particularly limited, but thawing can be performed, for example, in a hot water bath or water bath set to room temperature or the temperature planned for the culture process.

[0034] Any additional processing can be performed upon acquisition, for example, genome editing or genetic recombination of mesenchymal stem cells can be performed as needed.

[0035] Mesenchymal stem cells usable in the method of this embodiment may be in the form of a cell population containing mesenchymal stem cells. When a cell population is referred to by a cell type, it means that it contains cells of that type, and some of the cells may be of other types. Furthermore, when a cell population contains cells derived from multiple tissues, the name of one of the tissues can be used as a representative name for the cell population. For example, when a cell population is referred to as adipose tissue-derived mesenchymal cells, the cell population may contain adipose tissue-derived mesenchymal cells, but may also contain mesenchymal cells derived from other tissues or other cell types. In this case, the proportion of adipose tissue-derived mesenchymal cells is not particularly limited. For example, the proportion may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0036] 1-3-2.Culture process The culturing step is an optional step in the method of the present invention, and is a step of culturing mesenchymal stem cells. If an obtaining step is performed, this step can be performed after the obtaining step.

[0037] The purpose of the culture in this step is not particularly limited, but may be, for example, to grow the cells, to wake the cells, or to acclimate the cells.

[0038] The culture method in this step is not particularly limited. For example, it may be suspension culture or adhesion culture. For example, adhesion culture is preferable.

[0039] "Adhesion culture" is a cell culture method in which cells are cultured by adhering them to the surface of a culture vessel or the like. Typically, an external matrix or the like is used to adhere the cells to the surface of the culture vessel or the like, but if the cells are capable of adhering to the surface of the culture vessel, an external matrix need not be used. When an external matrix is ​​used, the specific type is not particularly limited. For example, laminin, vitronectin, gelatin, collagen, E-cadherin chimeric antibody, or a combination thereof can be used.

[0040] Furthermore, "suspension culture" refers to a cell culture method in which cells are cultured in a suspended state in a liquid medium. Here, "suspension state" refers to a state in which cells are not fixed by adhesion to an external matrix on the surface of a culture vessel or the like (e.g., the inner surface of the wall, bottom, underside of the lid, etc., or the surface of a structure within the culture vessel (e.g., agitator blades, etc.)). The method for suspending cells is not particularly limited, and examples include stirring, swirling, shaking, or a combination thereof. Furthermore, suspension culture in this specification also includes, for example, a culture method in which cells are attached to microcarriers and cultured in a suspended state in a culture medium.

[0041] The culture conditions used in this step are not particularly limited. For example, the culture conditions, such as cell seeding density, culture temperature, culture time, CO2 concentration, number of medium changes, number of passages, and detachment method, can be appropriately set depending on the purpose.

[0042] The seeding density of cells in the case of adherent culture is not particularly limited. 2 cells / cm 2 , 10 3 cells / cm 2 , 10 4 cells / cm 2 , 10 5 cells / cm 2 The upper limit can be set to, for example, 10 8 cells / cm 2 , 10 7 cells / cm 2 , 106 cells / cm 2 , 10 5 cells / cm 2 For example, 10 2 cells / cm 2 ~10 8 cells / cm 2 , 10 2 cells / cm 2 ~10 6 cells / cm 2 , 10 3 cells / cm 2 ~10 5 cells / cm 2 The cell density of the suspension culture or the cell concentration of the cell suspension used for seeding is not particularly limited. Specific lower limits include, for example, 10 2 cells / mL, 10 3 cells / mL, 10 4 cells / mL, 10 5 The upper limit can be, for example, 10 8 cells / mL, 10 7 cells / mL, 10 6 cells / mL, 10 5 cells / mL, etc. For example, 10 2 cells / mL ~10 8 cells / mL, 10 2 cells / mL ~10 6 cells / mL, 10 3 cells / mL ~10 5 A concentration of cells / mL can be suitably used.

[0043] The temperature during culture can be set appropriately and is not particularly limited. For example, the lower limit can be 25°C, 30°C, 32°C, or 35°C, and the upper limit can be 45°C, 40°C, or 38°C. Culture is usually carried out at about 37°C (35 to 38°C).

[0044] The culture period for one culture is not particularly limited, but can be, for example, 1 hour to 14 days, more specifically, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 24 hours or more, or, for example, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, or 2 days or less.

[0045] The CO2 concentration in the culture environment is not particularly limited. For example, the lower limit can be 0%, 1%, 2%, 3%, 4%, or 5%, and the upper limit can be 20%, 10%, 9%, 8%, 7%, 6%, or 5%. Typically, culture can be performed at a CO2 concentration of about 5% (3% to 7%).

[0046] The medium may be changed during the culture period. The medium may be changed multiple times, only once, or continuously (for example, by perfusion). When the medium is changed multiple times, the frequency of the medium change is not particularly limited. For example, it may be changed at predetermined intervals, or may be changed as needed in response to changes in the culture medium (for example, in response to changes in the pH of the culture medium). When the medium is changed at predetermined intervals, the frequency is not particularly limited. For example, it may be changed every 1 to 7 days, and specifically, it may be changed every 1 day, 2 days, 3 days, 4 days, 5 days, etc. The amount of medium changed in each change is not particularly limited. For example, all or half of the medium may be changed.

[0047] In this step, the cells can be passaged. The number of passages is not particularly limited. For example, the cells can be passaged once or more, twice or more, three or more, four or more, or five or more times. The number of passages may be, for example, 25 or less, 20 or less, 15 or less, or 10 or less.

[0048] The peeling means is not particularly limited. For example, the peeling means exemplified in the recovery step can be used.

[0049] The confluence rate at the end of the culture is not particularly limited, and examples include 1% or more, 10% or more, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, and 100%.

[0050] The medium used for culturing is not particularly limited as long as it is a medium in which mesenchymal stem cells can grow, and can be prepared by using any liquid medium as a basal medium and adding culture additives as needed. Examples of the other components include, but are not limited to, albumin, serum, serum replacement reagents, cytokines, antibiotics, etc. Two or more types of other components may be added in combination, or a commercially available medium containing multiple culture additives (e.g., serum-free medium) may be used.

[0051] Examples of basal media that can be used include, but are not limited to, common media such as BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)).

[0052] When the medium used in the method of this embodiment contains a culture additive, the specific type of culture additive is not particularly limited. As used herein, a "culture additive" refers to a substance other than a gaseous component that is added to a medium for the purpose of culture. Specific examples of culture additives include, but are not limited to, albumin, L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, or combinations thereof. Growth factors that can be used include, but are not limited to, basic fibroblast growth factor-2 (FGF2), transforming growth factor-β1 (TGF-β1), activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7, or combinations thereof. Antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, or a combination thereof. When a protein is used as a culture additive, it may be a naturally occurring protein isolated from the tissue or serum of an animal (e.g., a mammal such as a human, mouse, rat, cow, horse, or goat), or it may be a recombinant protein produced by genetic engineering, or a chemically synthesized protein.

[0053] When serum is contained, the type of serum is not particularly limited. The medium used in this step is preferably a serum-free medium. In this case, a serum replacement reagent can be used instead of serum. The specific type of serum replacement reagent is not particularly limited, but for example, platelet lysate (hPL) can be used.

[0054] As used herein, the term "platelet lysate" refers to a platelet-containing component obtained by lysing platelets, as well as a medium additive containing the platelet-containing component. Platelet lysate is a type of body fluid extract containing multiple platelet-containing components, such as growth factors (e.g., platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF2), and epidermal growth factor (EGF)) and cytokines. Platelet lysate preferably contains multiple platelet-containing components. The method for lysing platelets is not particularly limited, and examples include chemical treatment (e.g., treatment with CaCl2), physical treatment (e.g., physical disruption), osmotic pressure (e.g., exposure to a hypotonic solution such as water), and freeze-thawing. Platelet lysate prepared from animal body fluids may be used, or commercially available platelet cultures may be used directly or after processing. Examples of commercially available platelet lysates include NeoSERA (Kanto Chemical Co., Inc.), PLTMax Human Platelet Lysate (Merck), Human Platelet Lysate (STEMCELL Technologies), UltraGRO (AventaCell BioMedical), etc. The source of the platelet lysate is not particularly limited, and may be the same animal species as the source of the cell population containing mesenchymal cells, or a different animal species.

[0055] The form in which the culture additive is added to the medium is not particularly limited. For example, the culture additive can be added to the medium as is, or in the form of a solution, derivative, salt, mixed reagent, or the like.

[0056] The culture vessel used in this step is not particularly limited. For example, any culture vessel known in the art can be used. For example, dishes, multiwell plates (6-well plates, 12-well plates, 24-well plates, 96-well plates, etc.), culture chambers, roller bottles, culture flasks, shaker flasks, spinner flasks, etc. can be used. For example, in the case of suspension culture, culture flasks, shaker flasks, spinner flasks, etc. can be used, and in the case of adherent culture, dishes, multiwell plates, culture chambers, roller bottles, culture flasks, etc. can be used.

[0057] The material of the culture vessel used is not particularly limited. For example, a culture vessel made of glass or a resin such as polyethylene can be used. The inner surface of the culture vessel may be treated to allow or prevent cell adhesion, as necessary.

[0058] This step can be carried out multiple times. For example, it can be carried out once before the stimulation step described below, once simultaneously with the stimulation step, and once after the stimulation step. When this step is carried out multiple times, the culture conditions may be the same for all cultures, or two or more cultures may be carried out under different conditions.

[0059] During this step, the properties of the cells can be examined. The method for examination in this case is not particularly limited, but for example, the methods exemplified in the recovery step can be used.

[0060] 1-3-3. Stimulation process The stimulation step is an essential step in the method of the present invention, and is a step of irradiating mesenchymal stem cells with shock waves. When a culture step is performed, this step can be performed simultaneously with, before, and / or after the culture step. The stimulation step can be performed ex vivo or in vivo, as long as the conditions for irradiating mesenchymal stem cells with shock waves are met. Furthermore, this step can be performed once or multiple times. When performed multiple times, all irradiations may be performed under the same conditions, or two or more irradiations may be performed under different conditions.

[0061] The type of shock wave to be irradiated is not particularly limited, but is preferably an underwater shock wave. The manner of irradiating the shock wave is not particularly limited, but for example, a diverging shock wave or a converging shock wave may be irradiated.

[0062] The waveform of the irradiated shock wave is not particularly limited, but is preferably a square wave and / or a pulse wave.

[0063] The frequency of the shock waves to be applied is not particularly limited. For example, it can be 1,000 Hz or less, 500 Hz or less, 100 Hz or less, 50 Hz or less (low frequency), 25 Hz or less, 20 Hz or less, 15 Hz or less, 10 Hz or less, 9 Hz or less, 8 Hz or less, 7 Hz or less, 6 Hz or less, 5 Hz or less, or 4 Hz or less. Furthermore, the frequency can be, for example, 1 Hz or more, 2 Hz or more, 3 Hz or more, or 4 Hz or more. For example, frequencies of 1 Hz to 100 Hz, 1 Hz to 50 Hz, 2 Hz to 50 Hz, 2 Hz to 20 Hz, 3 Hz to 20 Hz, 3 Hz to 15 Hz, 4 Hz to 15 Hz, 2 Hz to 10 Hz, 3 Hz to 9 Hz, and 4 Hz to 9 Hz can be suitably used. The "frequency" of the shock waves refers to the number of times the shock waves are applied per second. For example, "the shock wave has a frequency of 4 Hz" means that the shock wave is emitted four times per second (for example, at intervals of 1 / 4 second (1 / frequency)).

[0064] Suitable shock waves include, for example, low-frequency pulse shock waves, where "low frequency" refers to a frequency of 50 Hz or less.

[0065] The method for generating shock waves is not particularly limited. Shock waves generated by any method known in the art can be used. Specifically, shock waves generated by, for example, underwater discharge, piezoelectric elements (piezo elements, etc.), electromagnetic vibration, electromagnetic acoustics, microexplosives, piston movement, or a combination thereof can be used.

[0066] When using focused shock waves, the focusing method is not particularly limited. For example, the generated shock waves can be focused to a specific area (focus or focal area) by arranging a reflecting mechanism or a generating source toward the specific area.

[0067] The type of shock wave irradiation device used for irradiation is not particularly limited. For example, any shock wave irradiation device used in the art can be used. Specifically, for example, a shock wave therapy device used for extracorporeal shock wave therapy can be preferably used. Examples of such shock wave therapy devices include the DUOLITH (R) SD1 ultra (Storz Medical), Epos Ultra (Dornier MedTech), and the like are available, and either can be suitably used in the method of this embodiment.

[0068] In the case of the focused type, the focal length (depth) is not particularly limited as long as it allows shock waves to be irradiated onto the cells and can be set appropriately depending on the distance between the outlet and the cells. For example, it can be 0.1 mm to 100 mm, 0.1 mm to 65 mm, 1 mm to 50 mm, 5 mm to 20 mm, 10 mm to 15 mm, etc. When shock waves are irradiated onto cells adhered to the inner wall of a container, the shock waves can be irradiated from inside the container, focusing on the inner wall.

[0069] The method of shock wave irradiation is not particularly limited, but preferably, the material from the outlet to the cells is made of a material capable of conducting shock waves. In this specification, "made of a material capable of conducting shock waves" means that the acoustic impedance under irradiation conditions is 1 MRayl (= 1 × 10 6 kg / (s·m 2The acoustic impedance range is not particularly limited, but may be, for example, 1 MRayl to 10 MRayl, 1.1 MRayl to 5 MRayl, 1.2 MRayl to 3.5 MRayl, 1.3 MRayl to 3.5 MRayl, 1.4 MRayl to 3.2 MRayl, 1.4 MRayl to 3 MRayl, 1.4 MRayl to 2.9 MRayl, 1.4 MRayl to 2.8 MRayl, 1.4 MRayl to 2.5 MRayl, 1.4 MRayl to 2.3 MRayl, etc.

[0070] There are no particular limitations on the method for measuring acoustic impedance. Acoustic impedance is a value specific to each material and can be determined based on information known in the art. For example, acoustic impedance may be measured using an acoustic impedance measuring device, or calculated by multiplying the density of the material by the sound velocity of the material.

[0071] The specific material that constitutes the space between the injection port and the cells is not particularly limited. Usable materials (acoustic impedance values ​​(MRayl)) include the following: water (approximately 1.48), culture medium (approximately 1.48), fat (approximately 1.4), blood (approximately 1.68), connective tissue (approximately 1.81), urethane rubber (approximately 1.31), polyurethane (approximately 1.66), acrylic gel (approximately 1.7), oil gel (approximately 1.54), gelatin (approximately 1.6), high-density polyethylene resin (approximately 2.04), polypropylene resin (approximately 2.25), ABS resin (approximately 2.26), polycarbonate resin (approximately 2.61), polyethylene terephthalate resin (approximately 2.87), acrylic resin (approximately 3.07), and rigid polyvinyl chloride resin (approximately 3.10). The above-mentioned materials or combinations thereof can be used in this process.

[0072] Furthermore, materials such as jelly, gel, and resin that are commonly used in ultrasound diagnosis and extracorporeal shock wave therapy can be used in this process without measuring the acoustic impedance.

[0073] In the irradiation of this step, it is preferable that a material with a significantly different acoustic impedance, such as air (acoustic impedance: approximately 0.0004 MRayl), does not intervene between the injection port and the cells. Also, it is preferable that the shock wave is irradiated so that no gas intervenes between the injection port and the cells.

[0074] When this process is performed ex vivo after the culturing process, it may be performed using a container or solution different from the container or medium used in the culturing process, or it may be performed without changing them. Alternatively, this process may be performed during the culturing process and then the culturing process may be continued. For example, shock waves can be irradiated by contacting the outlet of a shock wave irradiation device with the outer wall of the culture vessel. Alternatively, shock waves can be irradiated by contacting the outlet of a shock wave irradiation device with the culture medium or a liquid containing cells, or by contacting a material capable of transmitting shock waves with the surface of the culture medium or the liquid containing cells, and then contacting the outlet of the shock wave irradiation device directly or indirectly with the material. When using a container or solution different from the container or medium used in the culturing process, the type of container or liquid is not particularly limited. For example, the container may be any of the containers exemplified in the culturing process, or any other container known in the art. The liquid is not particularly limited as long as it does not kill the cells contained therein. Specific examples include buffer solutions, cell preservation solutions, cell cryopreservation solutions, and solvents for cell preparations exemplified in the third aspect. When this step is carried out in vivo, the cell preparation obtained after the culturing step, if necessary through steps such as recovery, freezing, and thawing, can be administered to a living body, and then the stimulation step can be carried out by irradiating shock waves from outside the body of the administered subject to the mesenchymal stem cells present in the living body. In this case, shock waves may be irradiated to the mesenchymal stem cells present in the living body multiple times at different angles.

[0075] The total energy of the stimulus applied in this step is not particularly limited. Specific lower limits for the total energy can be, for example, 0.005mJ / cell, 0.01mJ / cell, 0.011mJ / cell, 0.012mJ / cell, 0.013mJ / cell, 0.014mJ / cell, 0.015mJ / cell, 0.016mJ / cell, 0.017mJ / cell, 0.02mJ / cell, 0.03mJ / cell, 0.031mJ / cell, 0.032mJ / cell, 0.035mJ / cell, 0.04mJ / cell, 0.05mJ / cell, 0.06mJ / cell, 0.07mJ / cell, 0.08mJ / cell, 0.09mJ / cell, 0.094mJ / cell, 0.095mJ / cell, 0.096mJ / cell, etc. The upper limit is, for example, 0.5mJ / cell, 0.2mJ / cell, 0.19mJ / cell, 0.18mJ / cell, 0.17mJ / cell, 0.16mJ / cell, 0.15mJ / cell, 0.14mJ / cell, 0.13mJ / cell, 0.128mJ / cell, 0.125mJ / cell, 0.11mJ / cell, 0.1mJ / cell, 0.099mJ / cell, 0.098mJ / cell, 0.097mJ / cell, 0. The energy can be 0.096mJ / cell, 0.095mJ / cell, 0.09mJ / cell, 0.08mJ / cell, 0.07mJ / cell, 0.06mJ / cell, 0.05mJ / cell, 0.04mJ / cell, 0.035mJ / cell, 0.034mJ / cell, 0.033mJ / cell, 0.032mJ / cell, 0.03mJ / cell, 0.025mJ / cell, 0.02mJ / cell, 0.017mJ / cell, etc.For example, energies such as 0.005 mJ / cell to 0.2 mJ / cell, 0.01 mJ / cell to 0.17 mJ / cell, 0.015 mJ / cell to 0.17 mJ / cell, 0.016 mJ / cell to 0.16 mJ / cell, 0.015 mJ / cell to 0.15 mJ / cell, 0.015 mJ / cell to 0.13 mJ / cell, 0.016 mJ / cell to 0.128 mJ / cell, 0.015 mJ / cell to 0.12 mJ / cell, 0.015 mJ / cell to 0.1 mJ / cell, 0.015 mJ / cell to 0.09 mJ / cell, and 0.016 mJ / cell to 0.08 mJ / cell can be suitably used. For example, the energy may be changed depending on the gene to be enhanced. Although not particularly limited, for example, when enhancing a gene belonging to the TGF-β superfamily, the dose can be 0.015mJ / cell to 0.12mJ / cell, 0.02mJ / cell to 0.12mJ / cell, 0.025mJ / cell to 0.12mJ / cell, 0.026mJ / cell to 0.12mJ / cell, 0.03mJ / cell to 0.12mJ / cell, 0.035mJ / cell to 0.12mJ / cell, 0.04mJ / cell to 0.11mJ / cell, 0.05mJ / cell to 0.11mJ / cell, 0.06mJ / cell to 0.11mJ / cell, 0.06mJ / cell to 0.1mJ / cell, 0.07mJ / cell to 0.1mJ / cell, 0.07mJ / cell to 0.09mJ / cell, etc.For example, when enhancing a gene encoding interleukin, the following values ​​are used: 0.01mJ / cell to 0.17mJ / cell, 0.01mJ / cell to 0.16mJ / cell, 0.01mJ / cell to 0.15mJ / cell, 0.01mJ / cell to 0.13mJ / cell, 0.01mJ / cell to 0.12mJ / cell, 0.01mJ / cell to 0.1mJ / cell, 0.01mJ / cell to 0.09mJ / cell, 0.01mJ / cell to 0. The energy can be 0.08mJ / cell, 0.015mJ / cell to 0.08mJ / cell, 0.01mJ / cell to 0.07mJ / cell, 0.01mJ / cell to 0.05mJ / cell, 0.01mJ / cell to 0.035mJ / cell, 0.01mJ / cell to 0.03mJ / cell, 0.01mJ / cell to 0.025mJ / cell, 0.01mJ / cell to 0.02mJ / cell, 0.015mJ / cell to 0.02mJ / cell, etc.

[0076] The method for measuring the total energy of the stimulation applied in this step is not particularly limited. For example, it may be measured directly or calculated based on a formula. In this specification, the stimulation per irradiation [mJ / (cell impulse)] and the number of irradiations [impulse] applied to each cell satisfy the relationship of the following formula I. (Total stimulation given to each cell [mJ / cell]) = (Stimulus given to each cell per irradiation [mJ / (cell·impulse)]) × (Number of irradiations [impulse]) ...Equation I

[0077] Here, the method for measuring the stimulation per irradiation [mJ / (cell impulse)] given to each cell is not particularly limited. For example, it may be measured directly or calculated based on a formula. In this specification, the irradiation intensity per irradiation [mJ / (mm 2 impulse)] and irradiated cell density [cells / mm 2 ] satisfies the relationship of the following formula II. (Stimulus per irradiation given to each cell [mJ / (cell impulse)]) = (Irradiation intensity per irradiation [mJ / (mm 2 impulse)]) / (irradiation target cell density [cells / mm 2 ]) …Formula II

[0078] Here, the cell density to be irradiated [cells / mm 2 For example, when a focused shock wave is irradiated onto the culture surface, the seeding density [cells / mm 2 ] may be used as it is, and the irradiation target area [mm 2 ] and the number of cells to be irradiated [cells], it may be calculated based on the following formula III. (Irradiation target cell density [cells / mm 2 ]) = (number of cells to be irradiated [cells]) / (area of ​​the target area where cells are present [mm 2 ]) …Formula III

[0079] There are no particular limitations on the specific values ​​of the above items (number of irradiations, irradiation intensity per irradiation, density of cells to be irradiated, area of ​​the irradiation target where cells exist, number of cells to be irradiated).

[0080] Specific examples of the number of irradiations include a lower limit of 5, 10, 20, 25, 50, 75, 90, 100, 150, 200, 300, 400, and 500. Furthermore, an upper limit of 3000, 2000, 1000, 900, 750, 700, 600, 500, 400, 300, 250, 200, and 100. Suitable examples of irradiations include 5 to 1000, 50 to 600, 50 to 500, 50 to 400, 100 to 600, 100 to 500, 200 to 600, and 400 to 600. For example, the number of times may be changed depending on the gene to be enhanced. For example, when enhancing a gene belonging to the TGF-β superfamily, the number of times may be set to 150 or more, 200 or more, 300 or more, 400 or more, or 500 or more, without being particularly limited thereto.

[0081] Specific examples of the stimulation per irradiation given to each cell include lower limits of 0.05 μJ / (cell·impulse), 0.1 μJ / (cell·impulse), 0.15 μJ / (cell·impulse), 0.16 μJ / (cell·impulse), 0.17 μJ / (cell·impulse), 0.2 μJ / (cell·impulse), 0.25 μJ / (cell·impulse), 0.29 μJ / (cell·impulse), 0.3 μJ / (cell·impulse), 0.31 μJ / (cell·impulse), and 0.32 μJ / (cell·impulse). Also, for example, the upper limits are 5μJ / (cell·impulse), 2μJ / (cell·impulse), 1μJ / (cell·impulse), 0.9μJ / (cell·impulse), 0.7μJ / (cell·impulse), 0.65μJ / (cell·impulse). lse), 0.64μJ / (cell·impulse), 0.63μJ / (cell·impulse), 0.6μJ / (cell·impulse), 0.5μJ / (cell·impulse), 0.4μJ / (cell·impulse), 0.35μJ / (cell·impulse) impulse), 0.34μJ / (cell·impulse), 0.33μJ / (cell·impulse), 0.32μJ / (cell·impulse), 0.31μJ / (cell·impulse), 0.3μJ / (cell·impulse), 0.25μJ / (cell·impulse), 0.2μJ / (cell·impulse), 0.19μJ / (cell·impulse), 0.18μJ / (cell·impulse), 0.17μJ / (cell·impulse), 0.16μJ / (cell·impulse), etc.For example, 0.05μJ / (cell·impulse)~5μJ / (cell·impulse), 0.1μJ / (cell·impulse)~0.7μJ / (cell·impulse), 0.15μJ / (cell·impulse)~0.65 μJ / (cell·impulse), 0.16μJ / (cell·impulse)~0.64μJ / (cell·impulse), 0.15μJ / (cell·impulse)~0.6μJ / (cell·impulse), 0.15μJ / ( Suitable stimuli include 0.5 μJ / (cell impulse), 0.15 μJ / (cell impulse) to 0.4 μJ / (cell impulse), 0.15 μJ / (cell impulse) to 0.35 μJ / (cell impulse), 0.16 μJ / (cell impulse) to 0.32 μJ / (cell impulse), and 0.15 μJ / (cell impulse) to 0.3 μJ / (cell impulse). The strength of the stimulus may be varied depending on the gene to be enhanced. While not particularly limited, for example, when enhancing a gene belonging to the TGF-β superfamily, the strength may be 0.15 μJ / (cell impulse) to 0.25 μJ / (cell impulse), 0.15 μJ / (cell impulse) to 0.2 μJ / (cell impulse), etc.

[0082] The irradiation intensity per irradiation can be the energy flux density of the shock wave irradiation device used. Specific irradiation intensity per irradiation can be, for example, a lower limit of 0.005 mJ / (mm 2 impulse), 0.01mJ / (mm 2 impulse), 0.025mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse), 0.05mJ / (mm 2 impulse), 0.06mJ / (mm 2 impulse), 0.07mJ / (mm 2impulse), 0.08mJ / (mm 2 impulse), 0.09mJ / (mm 2 impulse), 0.1mJ / (mm 2 For example, the upper limit can be 5 mJ / (mm 2 impulse), 2mJ / (mm 2 impulse), 1mJ / (mm 2 impulse), 0.5mJ / (mm 2 impulse), 0.25mJ / (mm 2 impulse), 0.2mJ / (mm 2 impulse), 0.19mJ / (mm 2 impulse), 0.15mJ / (mm 2 impulse), 0.11mJ / (mm 2 impulse), 0.1mJ / (mm 2 impulse), 0.09mJ / (mm 2 impulse), 0.08mJ / (mm 2 impulse), 0.07mJ / (mm 2 impulse), 0.06mJ / (mm 2 impulse), 0.05mJ / (mm 2 For example, 0.005 mJ / (mm 2 impulse)~5mJ / (mm 2 impulse), 0.025mJ / (mm 2 impulse)~0.5mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.3mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.25mJ / (mm 2 impulse), 0.05mJ / (mm 2 impulse)~0.2mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.19mJ / (mm2 impulse), 0.04mJ / (mm 2 Impulse)~0.15mJ / (mm 2 impulse), 0.05mJ / (mm 2 impulse)~0.1mJ / (mm 2 The irradiation intensity may be changed depending on the gene to be enhanced. For example, when enhancing a gene belonging to the TGF-β superfamily, the irradiation intensity may be changed to 0.04 mJ / (mm 2 impulse)~0.09mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.08mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.07mJ / (mm 2 impulse), 0.04mJ / (mm 2 impulse)~0.06mJ / (mm 2 ·impulse) etc.

[0083] The density of cells to be irradiated can be set according to the culture conditions and cell suspension conditions, and is not particularly limited. A specific lower limit is, for example, 1 × 10 2 cells / mm 2 , 1×10 3 cells / mm 2 , 1×10 4 cells / mm 2 , 2 × 10 4 cells / mm 2 , 2.5×10 4 cells / mm 2 , 3×10 4 cells / mm 2 , 3.1 × 10 4 cells / mm 2 The upper limit can be set to, for example, 1×10 7 cells / mm 2 , 1×10 6 cells / mm 2, 1×10 5 cells / mm 2 , 9×10 4 cells / mm 2 , 5×10 4 cells / mm 2 , 4×10 4 cells / mm 2 For example, 1×10 2 cells / mm 2 ~1×10 7 cells / mm 2 , 1×10 3 cells / mm 2 ~1×10 6 cells / mm 2 , 1×10 4 cells / mm 2 ~1×10 5 cells / mm 2 , 2 × 10 4 cells / mm 2 ~1×10 5 cells / mm 2 , 2.5×10 4 cells / mm 2 ~1×10 5 cells / mm 2 , 2 × 10 4 cells / mm 2 ~9×10 4 cells / mm 2 , 3×10 4 cells / mm 2 ~5×10 4 cells / mm 2 , 3.1 × 10 4 cells / mm 2 ~4×10 4 cells / mm 2 The following cell densities can be suitably used.

[0084] The irradiation target area where cells exist is not particularly limited as long as it is the area (irradiation target surface) where shock waves are irradiated and where cells exist. For example, the area of ​​the culture surface, the bottom area of ​​the container, the area (irradiation target surface) where shock waves are irradiated, etc. can be used as the irradiation target area where cells exist. When converging shock waves are irradiated to cells suspended in liquid or cells present in a living organism, the area of ​​the focal region can be used as the irradiation target area. When diverging shock waves are irradiated to cells suspended in liquid or cells present in a living organism, the irradiation target area can be, for example, the area of ​​the focal region in the irradiation direction of the shock waves (for example, a cylindrical region extending forward from the outlet, with a cross-sectional area equal to a constant multiple of the area of ​​the outlet and a certain depth). (For example, in the above-mentioned cylindrical region, when shock waves are irradiated in a cone shape where the area is 0 at the outlet and the cross-sectional area of ​​the cylindrical region at the maximum depth, one-third of the cross-sectional area can be used as the irradiation target area.)

[0085] The number of cells to be irradiated is not particularly limited as long as it is the number of cells present on the surface to be irradiated. For example, when it can be assumed that all of the seeded or administered cells are present on the surface to be irradiated (for example, in the case of extracellular, all of the seeded cells are attached to the surface to be irradiated), the number of seeded cells or the number of administered cells can be used.

[0086] The specific combination of shock wave irradiation conditions (number of irradiations, frequency, amount of stimulation energy per cell, etc.) is not particularly limited. For example, the frequency may be 2 Hz to 10 Hz, the number of shock wave irradiations may be 100 to 600, and the total stimulation energy per cell may be 0.015 mJ to 0.17 mJ. Alternatively, the frequency may be 2 Hz to 10 Hz, the number of shock wave irradiations may be 400 to 600, and the total stimulation energy per cell may be 0.05 mJ to 0.1 mJ. Alternatively, the frequency may be 2 Hz to 10 Hz, the number of shock wave irradiations may be 400 to 600, and the total stimulation energy per cell may be 0.01 mJ to 0.1 mJ.

[0087] This step may be performed on cells in culture, or on cells suspended in a solution other than the medium or adhered to a container in a solution other than the medium. It may also be performed on cells administered into a living body as described above. Preferably, this step is performed on cells in culture.

[0088] 1-3-4. Recovery process The recovery step is an optional step in the method of the present invention, which is a step of recovering mesenchymal stem cells after culture, or, if the stimulation step is carried out ex vivo, a step of recovering mesenchymal stem cells after the stimulation step.

[0089] The method for recovering cells is not particularly limited. For example, when cells adhere to a container, they can be recovered by detaching the cells. Furthermore, when cells are suspended in a suspension, mesenchymal stem cells can be recovered by recovering the suspension.

[0090] The cells may be detached and dispersed by mechanical stimulation such as pipetting, or by chemical stimulation using the action of an enzyme such as trypsin and / or a chelating agent. The specific enzyme and chelating agent used are not particularly limited. Specific enzymes include trypsin, collagenase, pronase, hyaluronidase, elastase, and commercially available Accutase. (R) , Accumax (R) , TrypLE TM Express Enzyme (Life Technologies Japan, Inc.), TrypLE TM Select Enzyme (Life Technologies Japan, Inc.), Dispase (R) or a combination thereof. Specific examples of the chelating agent include EDTA, EGTA, or a combination thereof.

[0091] The method for recovering the suspension is not particularly limited. The suspension can be recovered by a conventional method, but can also be recovered by using methods used for removing culture supernatant, such as decantation and aspiration.

[0092] If necessary, contaminants such as cell fragments may be removed from the cell suspension by any method, including, but not limited to, centrifugation, filtration, or a combination thereof.

[0093] Furthermore, if desired, cells, mesenchymal stem cells or mesenchymal stem cells with enhanced expression of genes encoding cytokines can be enriched.

[0094] The method for concentrating cells used in this step is not particularly limited. Specifically, for example, methods using ultracentrifugation such as density gradient centrifugation, microfiltration, and ultrafiltration, methods using antibody capture such as immunoprecipitation, polymer precipitation, gel filtration, FACS, methods using microfluidic systems, size exclusion chromatography, HPLC, methods using lectins to adsorb onto carriers, methods using magnetic beads, or combinations thereof can be used.

[0095] The conditions for each method are not particularly limited as long as they allow the concentration of target cells, and can be appropriately selected based on the properties of the target cells, such as surface antigens and cell size.

[0096] For example, when using a method involving antibody capture, mesenchymal stem cells can be concentrated using, for example, an antibody capable of binding to the marker exemplified in the definition of mesenchymal stem cells in the first aspect.

[0097] The properties of the recovered cells can be confirmed by known methods, for example, by visually confirming them using an optical microscope or the like, by qualitatively or quantitatively confirming the amount of cytokine secreted using a solution containing secreted products from the cells, or by directly measuring the expression level of a gene encoding the cytokine.

[0098] The amount of cytokine secretion can be confirmed by any method capable of detecting a protein of interest in a solution, including, but not limited to, immunohistochemical analysis, Western blot analysis, immunoprecipitation, molecular binding assay, ELISA, flow cytometry, biochemical enzyme activity assay, etc.

[0099] The gene expression measurement method can be any method capable of detecting the transcription product of a gene of interest in a solution, and is not particularly limited. Examples include RT-PCR using primers or probes specific to the nucleotide sequence of the gene of interest, quantitative PCR, RNA-Seq using a sequencer, a method using a microarray (microchip), Northern blotting, or a combination thereof.

[0100] The cytokines detected in this step and the changes in their expression levels are as described in the second embodiment.

[0101] 1-4.Effects According to the method of this embodiment, it is possible to easily modify the properties of mesenchymal stem cells and enhance the expression of genes encoding cytokines without genetic modification, and the cells obtained by the method of this embodiment can achieve any desired effect based on the enhanced expression of cytokines.

[0102] 2.Cells 2-1. Overview A second aspect of the present invention relates to cells. The cells of the present invention are mesenchymal stem cells in which expression of a gene encoding a cytokine is enhanced, produced by the production method described in the first aspect (including cases in which expression of a gene encoding a cytokine is enhanced by a stimulating step in vivo). The cells of this aspect can be an active ingredient of the cell preparation described in the third aspect.

[0103] 2-2.Configuration The cells used in the present invention can be derived from any one or more organisms, such as those exemplified in the section on the obtaining step of the first aspect. For example, mammalian cells, human cells, cells derived from an organism of the same species as the subject, cells derived from an individual subject to the use, or a combination thereof can be used.

[0104] The cells of this embodiment may all be derived from the same tissue, organ, individual, or species, or may be a mixture of cells derived from multiple tissues, etc. The cells of this embodiment are preferably mesenchymal stem cells derived from fetal appendages, amniotic membrane, adipose tissue, or a combination thereof of a primate such as a human.

[0105] The individual and tissue from which the cells of this embodiment are derived may be suffering from a particular disease or may be healthy.

[0106] In the cells of this embodiment, expression of a gene encoding a cytokine is enhanced. The cytokines whose expression is enhanced in the cells of this embodiment are not particularly limited. Examples include interleukins, proteins belonging to the TGF-β superfamily, interferons (INFs), chemokines, tumor necrosis factors (TNFs), MCPs, etc. Preferred cytokines herein include, for example, proteins belonging to the interleukins and / or TGF-β superfamily.

[0107] The specific type of interleukin is not particularly limited and may be any of various interleukin family proteins. Examples include IL1 family proteins, IL2 family proteins, IL3 family proteins, and IL4 family proteins. For example, the expression of a gene encoding an IL1 family protein is preferably enhanced. Examples of IL1 family proteins include IL1α, IL1β, IL1RN, IL18, IL33, IL36, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, and IL1F10. In the cells of this embodiment, the expression of a gene encoding IL1RN is preferably enhanced, for example.

[0108] The specific type of protein belonging to the TGF-β superfamily is not particularly limited. For example, it may be a protein belonging to any of the TGF-β family, GDF family, GDNF family, inhibin family, activin family, and BMP family, or other proteins such as MIS, AMH, Lefty, and Nodal. Examples of TGF-β family proteins include TGF-β1, TGF-β2, and TGF-β3. In the cells of this embodiment, the expression of a gene encoding TGF-β1 is preferably enhanced, for example.

[0109] As used herein, "enhanced gene expression" refers to an increase in the expression level compared to mesenchymal stem cells not irradiated with shock waves, and the degree of this increase is not particularly limited. For example, the expression level may be significantly increased, and the expression level may be increased by 1.10-fold or more, 1.15-fold or more, 1.20-fold or more, 1.25-fold or more, 1.30-fold or more, 1.40-fold or more, 1.50-fold or more, 1.60-fold or more, 1.70-fold or more, 1.80-fold or more, 1.90-fold or more, 2.00-fold or more, 2.05-fold or more, 2.10-fold or more, 2.20-fold or more, 2.30-fold or more, 2.35-fold or more, 2.40-fold or more, 2.45-fold or more, 2.50-fold or more, 2.60-fold or more, 2.70-fold or more, or 2.80-fold or more compared to mesenchymal stem cells not irradiated with shock waves.

[0110] The method for measuring the expression level is not particularly limited. For example, the method exemplified in the recovery step of the first embodiment can be used. The method for comparing the expression level is not particularly limited. For example, in the case of a method based on PCR, the expression level can be standardized using the results of an internal standard and a control condition (condition without shock wave irradiation). -ΔΔCt The results may be determined based on the value of 2 -ΔΔCt If the value of is equal to or greater than a certain value, it may be determined that the expression of the gene is enhanced.

[0111] The expression level may be determined by measuring the amount of protein produced or secreted.

[0112] In this case, for example, the amount of protein (e.g., the amount of secreted TGF-β1) may be significantly increased, and the amount of protein may be increased by 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.25 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.5 times or more, 3.0 times or more, 3.1 times or more, 3.2 times or more, 3.3 times or more, 3.4 times or more, 3.5 times or more, 4.0 times or more, 4.5 times or more, 4.6 times or more, 4.7 times or more, 4.8 times or more, or 4.9 times or more compared to mesenchymal stem cells that have not been irradiated with shock waves.

[0113] It is preferable that the amount of expression of factors that promote inflammatory responses (IL-1, etc.) in mesenchymal stem cells is not increased, or is not significantly increased, or is not significantly increased, by shock wave irradiation.

[0114] The cells of this embodiment can be subjected to any additional treatment as needed. The specific treatment is not particularly limited, but can include, for example, genetic modification treatment or preservation treatment such as freezing.

[0115] The cells of this embodiment may be provided in the form of a cell population. In this case, the percentage of mesenchymal stem cells contained is not particularly limited. For example, the percentage may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0116] 3. Cell Preparation Overview A third aspect of the present invention is a cell preparation. The cell preparation of this aspect contains the cells described in the second aspect as an active ingredient. The cell preparation of the present invention can provide, in a subject, the effects of mesenchymal stem cells, as well as the effects of cytokines resulting from enhanced expression of genes encoding the mesenchymal stem cells.

[0117] 3-2.Configuration The cell preparation of this embodiment contains the cells according to the second embodiment as essential components, and optionally contains solvents and additives. Each component will be specifically described below.

[0118] 3-2-1.Cells The cells contained in the cell preparation of this embodiment are as described in the second embodiment. The biological species from which the cells contained in the cell preparation of this embodiment are derived is not particularly limited. For example, the cells may be from the biological species or individual to be administered, or may be from the biological species or individual to be treated.

[0119] The cells used in the cell preparation of this embodiment can be subjected to any treatment before contacting them with the preservation solution. Specific treatments are not particularly limited, but examples include freezing, thawing, culturing, recovery, washing, selection, transformation, genetic manipulation, or a combination thereof.

[0120] The state of cells in the cell preparation of this embodiment may be a single cell state or a cell aggregate state such as a spheroid. A single cell state is preferred. As used herein, "single cell state" refers to a state in which cells exist singly and are not aggregated. The proportion of single cells among all cells in the cell preparation is, for example, 70% or more, 90% or more, 95% or more, 99% or more, or 100%. The proportion of single cells in the cell preparation can be measured using any method. For example, the proportion can be measured by dispersing cells in a buffer solution (e.g., PBS) and observing randomly selected cells under a microscope to determine whether or not they aggregate.

[0121] The cells in the cell preparation may be floating or in contact with the inner wall of the container, etc. Preferably, the cells are floating. As used herein, "floating" refers to the cells not being fixed to the inner wall of the container containing the cell preparation by adhesion or the like. For example, the proportion of floating cells among all cells in the cell preparation is, for example, 40% or more, 50% or more, 60% or more, 70% or more, 90% or more, 95% or more, 99% or more, or 100%.

[0122] The cells in the cell preparation are, for example, 500 to 10,000 cells / cm 2 The cells were seeded onto the culture substrate at a density of 20,000 cells / cm. 2 The cells can be prepared by a method comprising culturing the cells until the temperature exceeds 100°C, immersing the cells in a trypsin solution at 20 to 40°C for 5 minutes or more to recover the cells, slowly cooling the cells at a rate of -5°C / min or less using a cryopreservation solution containing a cryoprotectant to freeze them, and then rapidly thawing the cells in a thermostatic chamber preheated to 25 to 42°C.

[0123] The number of cells contained in the cell preparation of the present invention per unit dose is not particularly limited. Generally, the number of cells varies depending on the type of cells, the route of administration, the purpose of administration, and the type of carrier, which is another component described below. Therefore, it may be determined appropriately taking into consideration each condition. For example, it is sufficient that a sufficient number of cells is contained in a single dose of the cell preparation. The specific number of cells per unit dose is not particularly limited, but may be, for example, 1 x 10 3 ~1×10 11 1 × 10 4 ~1×10 10 pieces / mL, 1×10 5 ~1×10 9 pieces / mL, 1×10 6 ~1×10 8 The concentration can be, for example, cells / mL. When administered in multiple doses, the total amount should contain a sufficient number of cells. When the cell preparation of the present invention is diluted and administered, the diluted preparation should contain a sufficient number of cells to achieve the desired effect.

[0124] Any cells can be additionally included in addition to the cells described in the second aspect. The type of additional cells is not particularly limited. Preferably, the cells are derived from the same species or the same individual as the cells described in the second aspect.

[0125] 3-2-2.Solvent The cell preparation of this embodiment may contain a pharmaceutically acceptable solvent as needed. The term "pharmaceutically acceptable solvent" refers to a solvent commonly used in the pharmaceutical technology field. Examples include water and aqueous solutions. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include inorganic salts such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and organic acid salts such as citrate, gluconate, and succinate, as well as low-concentration nonionic surfactants and polyoxyethylene sorbitan fatty acid esters.

[0126] The pH of the solution is not particularly limited as long as the cells to be preserved remain viable. Specific pH values ​​are, for example, 3.5 to 8.5, 4 to 8, 4.5 to 7.5, or 5 to 7.5.

[0127] The osmotic pressure of the solution is not particularly limited as long as it allows cells to survive. For example, the solution may be hypotonic (less than 250 mOsm / L), isotonic (250 mOsm / L to 380 mOsm / L), or hypertonic (more than 380 mOsm / L). The osmotic pressure may be expressed as the osmotic pressure ratio to physiological saline (e.g., 306 mOsm / L).

[0128] The solution of the present invention may be prepared by the patient himself or may be commercially available. For example, preferred solutions include Ringer's solution (lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, etc.), Ringer's basal solution, and any other solution used as an infusion solution.

[0129] The viscosity of the solution is not particularly limited as long as the cells to be preserved remain viable. For example, at the temperature during cell suspension and / or storage, the viscosity is preferably 7 mPa·s or more, or 8 mPa·s or more, or 9 mPa·s or more, and preferably 18 mPa·s or less, or 15 mPa·s or less, or 13 mPa·s or less. The viscosity of the non-cryopreservation solution can be measured, for example, using a TV-20 viscometer (Toki Sangyo Co., Ltd.) at a rotation speed of 10 rpm.

[0130] 3-2-3.Additives The cell preparation of the present invention may contain pharmaceutically acceptable additives as needed. "Pharmaceutically acceptable additives" refer to additives commonly used in the pharmaceutical technology field. Examples include excipients, binders, disintegrants, emulsifiers, flow regulators, lubricants, etc.

[0131] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, etc. (R) , kaolin, silicic acid, or a combination thereof.

[0132] Examples of binders include starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, and combinations thereof.

[0133] Disintegrants include, for example, the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

[0134] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0135] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0136] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizing agents, humectants, humectants, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, buffers, pH adjusters, isotonic agents, and the like that are commonly used in pharmaceutical compositions, cell preparations, and the like.

[0137] Additives are used to avoid or inhibit the decomposition of the active ingredient by enzymes and the like in the subject's living body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0138] 3-3.Dosage form etc. The cell preparation of the present invention is in the form of a liquid. Liquid preparations include any preparations that have fluidity. Specific examples include creams, ointments, gels, injections, suspensions, etc. Specific volumes and the like are not particularly limited as long as they are within the range known in the art for each dosage form. The cell preparation of the present invention may be produced by a method commonly used in the art.

[0139] The cell preparation of the present invention is preferably in a sterile state. The method for achieving a sterile state is not particularly limited and any method known in the art can be used.

[0140] 3-4. Application method The cell preparation of the present invention can be administered parenterally. Parenteral administration can be further divided into systemic administration and local administration. Local administration includes, for example, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrathecal, intralesional, intracranial, intraosseous, tissue administration, and organ administration. Systemic parenteral administration includes intracirculatory administration (e.g., intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration), intraperitoneal administration, rectal administration, intranasal administration, intrabuccal administration, and intravaginal administration. For example, the cell preparation of the present invention can be administered locally, in which case it can be directly administered to the target site (including the oral cavity) by, for example, injection. Furthermore, the cell preparation can be incorporated into, for example, a cream, ointment, gel, suspension, or any other suitable substance at the time of application. Systemic administration can be performed, for example, by intracirculatory administration, such as intravenous injection. The dosage may be any amount effective for the cells to be effective. The effective amount is selected appropriately depending on the subject's information, as described above. For example, when using cells with homing ability (e.g., mesenchymal stem cells, etc.), even if the purpose is to administer them to a specific site, the cells may be delivered to the target site by simple systemic administration. Prior to administration, additional treatment, such as irradiation with shock waves, may be performed on the application site. In this case, the mesenchymal stem cells contained in the cell preparation may be mesenchymal stem cells that have been subjected to shock wave irradiation treatment, or may be mesenchymal stem cells that have not been subjected to shock wave irradiation treatment.

[0141] The application method and dosage of the cell preparation of the present invention may vary depending on information about the subject. As used herein, "subject information" refers to various information about the characteristics and condition of the subject. For example, when the subject is a human individual, information may include age, weight, sex, general health condition, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0142] The cell preparation of the present invention can be stored before application. The specific storage method, storage period, and storage temperature are not particularly limited.

[0143] Furthermore, the cell preparation of the present invention may be applied as is, or may be applied after any additional treatment. Specific additional treatments include, but are not limited to, application by transplantation of a reservoir containing the cell preparation of the present invention, or application by transplantation of tissues, organs, or organoids formed by further culturing the cell preparation. Further culturing can be performed for any purpose, such as increasing the number of cells, cell differentiation, transformation, or gene transfer into cells.

[0144] Furthermore, the cell preparation of the present invention can be used in combination with one or more known drugs and / or cell preparations. The types of drugs and / or cell preparations used in combination are not particularly limited. When the cell preparation of the present invention is a cell preparation for preventing or treating a specific disease or condition, it can be used in combination with one or more known drugs for preventing or treating that disease or condition, and / or one or more known drugs for preventing or treating other diseases or conditions. The drugs for preventing or treating symptoms or diseases that can be used in combination are not particularly limited. They can be selected appropriately depending on the condition of the patient and the type of disease or condition to be treated or prevented.

[0145] 3-5.Applicability There are no particular limitations on the application of the cell preparation of the present invention, and it can be applied to tissues, organs, or individuals of the organisms exemplified as species from which the mesenchymal stem cells of the first embodiment are derived (e.g., mammals, including primates such as humans, laboratory animals, and livestock).

[0146] As used herein, the term "subject" refers to a target to which the cell preparation of this embodiment is applied. For example, it is a tissue, an organ, or an individual. In the case of an individual, it is preferably a human individual. In the present invention, the subject may be healthy or afflicted with a disease, and includes, for example, an individual with inflammation or an individual expected to develop inflammation in the future. The presence or absence of inflammation can be determined by, for example, an increase in the production or activity of proteins related to inflammation or immune response, or the presence or absence of inflammatory symptoms.

[0147] As used herein, "subject information" refers to various information relating to the characteristics and condition of a subject. For example, when the subject is a human individual, information includes age, weight, sex, general health condition, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant medication, and resistance to treatment.

[0148] The purpose of application is not particularly limited. For example, they can be used for purposes such as the improvement, treatment, and prevention of diseases and conditions. Furthermore, for example, they can be used to enhance the effects of mesenchymal stem cells in therapies that typically use mesenchymal stem cells. In this case, the diseases or conditions for which mesenchymal stem cells are used are not particularly limited, and examples include cancer, leukemia, vascular diseases, stem cell exhaustion diseases, bone diseases, cartilage diseases, ischemic diseases, neurological diseases, burns, acute inflammation, chronic inflammation, heart diseases such as ischemic cardiomyopathy and dilated cardiomyopathy, immune diseases (Sjögren's syndrome, dermatomyositis, polymyositis, etc.), immunodeficiency, Crohn's disease, diabetes, arthropathy, facial lipoatrophy, mastectomy, scars, age spots, wrinkles, sagging skin, etc. Specific purposes include, for example, regenerative medicine for tissue augmentation in tissue depressions and the like, or for the treatment of osteoarthritis of the knee, immunotherapy such as T cell therapy, NKT cell therapy, and dendritic cell transfer therapy, gene therapy using gene-transferred cells, breast augmentation, breast reconstruction, wrinkle removal, age spot removal, and other cell transplant therapies.

[0149] The cell preparation of this embodiment may be applied for the purpose of suppressing inflammation. In this case, the cell preparation of this embodiment is used as an anti-inflammatory agent. Furthermore, for example, the cell preparation of this embodiment may be applied for the purpose of regulating the function of immune cells such as T cells and B cells. In this case, the cell preparation of this embodiment is used as an immunomodulator.

[0150] As used herein, the term "anti-inflammatory agent" refers to a substance that has the effect of preventing, treating, or alleviating inflammation that occurs in the body.

[0151] The term "inflammatory disease" refers to a disease that involves persistent or transient inflammation, and whose symptoms can be improved by reducing the inflammation. In this specification, inflammatory diseases include both chronic inflammatory diseases and acute inflammatory diseases. In this specification, inflammatory diseases include inflammatory symptoms including local or systemic primary symptoms caused by inflammatory cells activated by inflammation, and secondary symptoms induced thereby.

[0152] Inflammatory diseases to which the anti-inflammatory agent of the present invention can be applied include, for example, allergic inflammatory diseases such as delayed-type allergies; cancers caused by chronic inflammation such as colon cancer, lung cancer, bladder cancer, oral cancer, tongue cancer, skin cancer, esophageal cancer, melanoma, bile duct cancer, colon cancer, gallbladder cancer, stomach cancer, cervical cancer, and liver cancer; inflammatory diseases involving macrophages such as hepatitis (alcoholic hepatitis, non-alcoholic hepatitis, etc.), asthma, systemic lupus erythematosus, Crohn's disease, arthritis (chronic rheumatoid arthritis, etc.), osteoarthritis, bedsores, and dementia; arteriosclerosis, gastric ulcer, nephritis (glomerulonephritis, IgA nephropathy, diabetic nephropathy, etc.), and other conditions. Other inflammatory diseases include nephropathy, periodontal disease (gingivitis, peridontitis, etc.), liver cirrhosis, bronchitis, aneurysm, endometriosis, acute respiratory distress syndrome, kidney transplant injuries, acute myocardial infarction, diabetes, Crohn's disease, pneumonia, endotoxin shock, sepsis due to infection, chronic ulcerative colitis, chronic bronchitis, cystitis, chronic osteomyelitis, reflux esophagitis, cholangitis, chronic cholecystitis, gastritis, chronic cervicitis, celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel syndrome, atherosclerosis, ankylosing spondylitis, colitis, chronic active hepatitis, gastrointestinal stenosis, fistula, dermatitis, and psoriasis.

[0153] As described above, the present invention also provides a method for enhancing the expression of genes encoding cytokines in mesenchymal stem cells in vivo by administering a cell preparation containing mesenchymal stem cells to a living body and then irradiating the mesenchymal stem cells in vivo with shock waves. That is, a cell preparation in which the administration of the cell preparation and a stimulation step of irradiating the living body with shock waves are combined to enhance the expression of genes encoding cytokines in mesenchymal stem cells in vivo is also an aspect of the present invention. Another aspect of the present application is, for example, a cell preparation containing mesenchymal stem cells for use in enhancing the expression of genes encoding cytokines in the mesenchymal stem cells in vivo by administering the cell preparation and then irradiating the living body with shock waves. The application of the cell preparation in this case is not particularly limited, and the cell preparation can be used as a therapeutic agent for the diseases described above (e.g., an anti-inflammatory agent), but is preferably used, for example, as a therapeutic agent for osteoarthritis (e.g., an anti-inflammatory agent). That is, a therapeutic agent for osteoarthritis containing mesenchymal stem cells, characterized by combining administration to a joint site with irradiation of the joint site with shock waves, is also an aspect of the present invention. Another aspect of the present application is a therapeutic agent for osteoarthritis (e.g., an anti-inflammatory agent) comprising mesenchymal stem cells, for use in enhancing in vivo expression of a gene encoding a cytokine in the mesenchymal stem cells by administering the cell preparation to a joint site and then irradiating the site with shock waves. The therapeutic agent for osteoarthritis may also be provided as a therapeutic agent for osteoarthritis pain. When the cell preparation of the present invention is used as a therapeutic agent for osteoarthritis, the cell preparation is typically administered to a joint site of a human suffering from pain due to osteoarthritis. The joint site may be administered either intra-articularly or intra-bone near the joint. Furthermore, the frequency of shock wave irradiation is not particularly limited.

[0154] By combining the administration of a cell preparation with a stimulation step of irradiating shock waves to the living body, pain symptoms can be alleviated or improved compared to the effects observed after either shock wave irradiation treatment alone without administration of the cell preparation or administration of a cell preparation (therapeutic drug) without shock wave irradiation treatment. For example, irradiating the application site with shock waves before administration of the cell preparation (which can generally be expected to result in the accumulation of mesenchymal stem cells at the irradiated site) and then irradiating shock waves again after administration of the cell preparation is acceptable as a preferred embodiment. When shock wave irradiation is performed on the application site before or after administration, the mesenchymal stem cells contained in the therapeutic drug administered after shock wave irradiation are preferably those obtained by the above-described culturing, recovery, freezing, thawing, or a combination thereof (i.e., mesenchymal stem cells that have not been subjected to ex vivo shock wave irradiation treatment). The preferred cell concentration in the cell preparation, the viscosity of the cell preparation, and other additives are also as described above.

[0155] Effects The cell preparation of this embodiment can be effective against severe inflammatory symptoms that could not be addressed with conventional cell preparations based on mesenchymal stem cells, and can also be effective in alleviating and improving the inflammatory symptoms of osteoarthritis (e.g., knee osteoarthritis) and pain symptoms associated with inflammation. [Example]

[0156] Example 1: Analysis of the effect of shock wave irradiation treatment on cell characteristics (1) (the purpose) We investigate the effect of shock wave irradiation treatment on the properties of mesenchymal stem cells. (method) 1. Obtaining Mesenchymal Stem Cells Adipose tissue obtained from a healthy human individual was enzymatically treated with Collagenase (Worthington) and then cultured at 37°C in a 5% CO environment for 6 days to obtain a cell population containing adipose mesenchymal stem cells. The medium used for this was the same as the suspension medium described below.

[0157] The obtained mesenchymal stem cells were stored in a cell preservation solution (CP-1 (R)The cells were suspended in a solution of 25% human serum albumin and physiological saline (Kyokuto Pharmaceutical Co., Ltd.) at a ratio of 2:1:3, slowly frozen to -80°C, and then frozen and stored under liquid nitrogen.

[0158] 2.Surface antigen analysis The obtained adipose mesenchymal stem cell-containing cell population was confirmed to contain mesenchymal stem cells (CD73, CD90, and CD105 positive; CD31, CD34, and CD45 negative) by analysis of each surface antigen (CD73, CD90, CD105, CD31, CD34, and CD45) using a flow cytometer.

[0159] 3. Preculture Cryopreserved mesenchymal stem cells were thawed in a 37°C water bath and suspended in 40 mL of suspension medium (αMEM containing human platelet lysate (hPL) at a final concentration of 5%). The suspension was centrifuged at 800 × g for 5 minutes, and the supernatant was removed to recover the mesenchymal stem cells. The recovered mesenchymal stem cells were collected at a concentration of 1 × 10 5 The cells were suspended in a suspension medium to a cell density of 100 cells / mL to prepare a suspension for seeding. 100 μL of the suspension for seeding was added to each well of a 96-well plate (bottom area: 32 mm 2 ) and cultured for 24 hours at 37°C in a 5% CO environment. During this culture, the wells in which the cells were cultured were not adjacent to each other.

[0160] 4.Shock wave irradiation treatment After 24 hours of pre-culture, all of the culture medium was removed from each well. Then, each well was filled with the above-mentioned suspension medium and covered with a polypropylene (PP) plate seal (Stem Co.). The outlet of a focused shock wave irradiation device was placed so that it was in contact with the plate seal, and shock waves were irradiated to the cells at the bottom of each well. The shock wave irradiation device used was DUOLITH (R) SEPIA included with SD1 ultra (Storz Medical) (R) A handpiece was used. The focal depth was set to 15 mm to match the height of the well, and the frequency was set to 4 Hz. Other irradiation conditions for each condition are shown in Table 1 below.

[0161] [Table 1]

[0162] For each condition, the stimulation per irradiation and the total stimulation applied to each cell were calculated. Each value was calculated using the following formulas I, II, and III. In this case, the number of cells to be irradiated was 1 x 10 cells seeded in each well. 5 The area to be irradiated with cells was 32 mm2, which is the bottom area of ​​the well. 2 was calculated using (Irradiation target cell density [cells / mm 2 ]) = (number of cells to be irradiated [cells]) / (area of ​​the target area where cells are present [mm 2 ]) …Formula III (Stimulus per irradiation given to each cell [mJ / (cell impulse)]) = (Irradiation intensity per irradiation [mJ / (mm 2 impulse)]) / (irradiation target cell density [cells / mm 2 ]) …Formula II (Total stimulation given to each cell [mJ / cell]) = (Stimulus given to each cell per irradiation [mJ / (cell·impulse)]) × (Number of irradiations [impulse]) ...Equation I

[0163] The values ​​calculated for each condition are shown in Table 2. The unit of stimulation per irradiation given to each cell was converted to [μJ / (cell impulse)].

[0164] [Table 2]

[0165] After irradiation, the plate seal was removed, and 100 μL of medium was removed from each well, followed by culturing at 37° C. in a 5% CO 2 environment for 24 hours.

[0166] 5.RT-PCR analysis After incubation, remove all culture medium from all wells of each plate, wash with PBS, and then RNeasy (R) Total RNA was extracted from the cells using the Plus Mini Kit (QIAGEN). TM cDNA for RT-PCR analysis was synthesized using an RT reagent kit (Takara Bio Inc.) RNA extraction and cDNA synthesis were performed according to the manufacturer's protocol.

[0167] Based on the cDNA solution, qPCR TaqMan Gene Expression Assays were used to amplify the IL1RN gene sequence, TGF-β1 gene sequence, and GAPDH gene (internal control). (R) PCR was performed using a PCR primer set (Thermo Fisher Scientific). The PCR reaction consisted of holding at 50°C for 2 minutes, followed by a denaturation reaction at 95°C for 20 seconds, followed by 40 cycles of a denaturation reaction at 95°C for 1 second and an annealing reaction at 60°C for 20 seconds. Three samples (3 wells) were used for each condition. Primers used were qPCR TaqMan Gene Expression Assays (R) The attached primers for IL1RN, TGF-β1, and GAPDH were used.

[0168] (result) The results are shown in Figures 1 and 2. FIG. 1 shows the results of IL1RN gene expression, and FIG. 2 shows the results of TGF-β1 gene expression.

[0169] In the mesenchymal stem cells subjected to shock wave irradiation treatment (conditions B to E), a general tendency for gene expression to be enhanced was observed for both the IL1RN gene and the TGF-β1 gene.

[0170] In particular, shock wave irradiation increased the expression level of the IL1RN gene by approximately 2- to 2.8-fold (Figure 1B). Furthermore, the expression level of the TGF-β1 gene was also increased under each irradiation condition, with condition C in particular increasing the gene expression level by approximately 1.6-fold (Figure 2B).

[0171] These findings suggest that the anti-inflammatory effects of mesenchymal stem cells can be enhanced by irradiating them with extracorporeal shock waves.

[0172] Example 2: Analysis of the effect of shock wave irradiation treatment on cell characteristics (2) (the purpose) We investigate the effect of shock wave irradiation treatment on the properties of mesenchymal stem cells. (method) Obtaining mesenchymal stem cells, analyzing surface antigens, and pre-culture were performed as described in Example 1. Shock wave irradiation treatment and subsequent culture were performed in the same manner as in Example 1, except that they were performed under the conditions shown in Table 3.

[0173] [Table 3]

[0174] After incubation, the entire culture medium was collected from all wells of each plate, and the amounts of TGF-β1 and IL-1 proteins in the collected culture medium were measured using ELISA kits. The ELISA kits used were as follows. The amount of each protein was measured according to the protocol provided by the manufacturer. For TGF-β1 measurement: Human / Mouse / Rat / Porcine / Canine TGF-beta 1 ELISA Kit (R&D SYSTEMS) For IL-1 measurement: Human IL-1 beta / IL-1F2 ELISA Kit (R&D SYSTEMS)

[0175] (result) In the culture medium of mesenchymal stem cells treated with shock waves (conditions D' and D"), the amount of TGF-β1 was significantly increased under both conditions compared to mesenchymal stem cells not treated with shock waves (condition A: approximately 91.5 pg / mL) (condition D': approximately 3.4 times; condition D": approximately 4.9 times).

[0176] Furthermore, IL-1, which, unlike the IL1RN gene, is a factor that promotes inflammatory responses, did not significantly change the amount of IL-1 contained endogenously in the basal medium, regardless of whether shock wave irradiation treatment was performed or not.

[0177] These findings suggest that irradiating mesenchymal stem cells with extracorporeal shock waves enhances the anti-inflammatory effects of mesenchymal stem cells without causing side effects that induce inflammation.

Claims

1. A method for producing mesenchymal stem cells in which expression of a gene encoding a cytokine is enhanced, the method comprising a stimulating step of irradiating the mesenchymal stem cells with shock waves.

2. The method according to claim 1 , further comprising a culture step of culturing the mesenchymal stem cells.

3. The method of claim 2, wherein the culture is an adherent culture.

4. 2. The method of claim 1, wherein the frequency of the shock wave irradiation is 50 Hz or less.

5. The method according to claim 1, wherein the number of shock wave irradiations is in the range of 5 to 3000.

6. The method according to claim 1, wherein the stimulation applied in the stimulation step is a stimulation of 0.005 mJ to 0.2 mJ per cell in total.

7. The method according to claim 6, wherein the frequency of the shock waves is 2 Hz to 10 Hz, the number of times the shock waves are applied is 100 to 600, and the stimulation is a total stimulation of 0.015 mJ to 0.17 mJ per cell.

8. The method according to claim 7, wherein the shock wave is applied 400 to 600 times, and the stimulation is a total stimulation of 0.05 mJ to 0.1 mJ per cell.

9. The method of claim 1, wherein the cytokine comprises a TGF-β family protein and / or an interleukin.

10. The method of claim 1 , further comprising a recovery step of recovering mesenchymal stem cells after the stimulation step.

11. A cell produced by the method according to any one of claims 1 to 10.

12. A cell preparation comprising the cells of claim 11.

13. An anti-inflammatory agent comprising the cells of claim 11.

14. A method for enhancing the expression of a gene encoding a cytokine in a mesenchymal stem cell ex vivo, the method comprising a stimulating step of irradiating the mesenchymal stem cell with shock waves.

15. A cell preparation that enhances the expression of genes encoding cytokines in mesenchymal stem cells in vivo, wherein the administration of the cell preparation is combined with a stimulating step of irradiating the living body with shock waves.

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