Method for preparing cell for suppressing inflammation, cell for suppressing inflammation, Anti-inflammatory composition, and method for promoting inflammation suppression action

Abstract

The purpose of the present invention is to provide a cell having a higher therapeutic effect against inflammation.　The present invention is a method for preparing cells for suppressing inflammation, the method being characterized in that an ultrasonic treatment is performed on cells. In the ultrasonic treatment, it is preferable that the intensity of the ultrasonic waves is 50 mW / cm2 to 5000 mW / cm2, the frequency is 20 kHz to 20 MHz, and the cells are mesenchymal stem cells.

Description

Method for preparing inflammation-suppressing cells, inflammation-suppressing cells, anti-inflammatory composition, and method for promoting inflammation-suppressing activity

[0001] The present invention relates to a method for preparing inflammation-suppressing cells, inflammation-suppressing cells, an anti-inflammatory composition, and a method for promoting inflammation-suppressing activity.

[0002] Mesenchymal stem cells (MSCs) are multipotent progenitor cells that were first isolated from bone marrow by Friedenstein in 1982 (see Non-Patent Document 1). Mesenchymal stem cells have been shown to exist in various tissues, such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Non-Patent Documents 1 and 2). Since it is known that cells with functions equivalent to those of mesenchymal stem cells exist in the stromal cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc., mesenchymal stem cells are sometimes referred to as stromal cells.

[0003] It is well known that mesenchymal stem cells are used in regenerative medicine and the like due to their tissue repair and immunosuppressive effects. However, the mechanism by which mesenchymal stem cells exert their effects has not yet been fully elucidated, and few mesenchymal stem cells have been proven effective in clinical applications. In order to further enhance the therapeutic effects of mesenchymal stem cells, a method of treating mesenchymal stem cells with ultrasound has been developed (Patent Document 1). Patent Document 1 shows that treating cells with ultrasound can improve their proliferation and migration abilities. Under these circumstances, there is a demand for technology that can further enhance the therapeutic effects of mesenchymal stem cells.

[0004] International Publication No. 2023 / 149351

[0005] Pittenger F. M. et al. Science, (1999), 284, pp. 143-147Cell Transplantation, (2016), 25, pp. 829-848

[0006] In the circumstances described above, an object of the present invention is to provide cells that have a stronger therapeutic effect against inflammation.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in significantly enhancing the anti-inflammatory effect of cells by treating the cells with ultrasound, thereby completing the present invention. According to the present invention, cells with a higher therapeutic effect against inflammation can be provided. That is, the gist of the present invention is as follows.

[0008] [1] A method for preparing cells for inflammation suppression, comprising subjecting cells to ultrasonic treatment. [2] The ultrasonic intensity in the ultrasonic treatment is 50 mW / cm ２ ~5000mW / cm ２ [3] The method for preparing anti-inflammatory cells according to [1], wherein the frequency of the ultrasonic treatment is 20 kHz to 20 MHz. [4] The method for preparing anti-inflammatory cells according to any one of [1] to [3], wherein the cells are mesenchymal stem cells. [5] The method for preparing anti-inflammatory cells according to [4], wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells. [6] The method for preparing anti-inflammatory cells according to any one of [1] to [5], wherein the ultrasonic treatment step is performed once or multiple times. [7] The method for preparing anti-inflammatory cells according to [6], which does not include a step of culturing the cells after the ultrasonic treatment. [8] The method for preparing anti-inflammatory cells according to [7], wherein the final ultrasonic treatment step among the ultrasonic treatments is performed immediately before administering the cells to a subject. [9] The method for preparing anti-inflammatory cells according to any one of [1] to [8], wherein the ultrasonic treatment is performed on cells in a suspended state.

[10] Inflammation-suppressing cells prepared by the preparation method according to any one of [1] to [9].

[11] An anti-inflammatory composition containing the inflammation-suppressing cells according to

[10] .

[12] A method for promoting the anti-inflammatory effect of the cells, comprising subjecting the cells to ultrasonic treatment.

[13] The ultrasonic intensity in the ultrasonic treatment is 50 mW / cm. ２ ~5000mW / cm ２

[14] The method for promoting an anti-inflammatory effect according to

[12] , wherein the frequency of the ultrasonic treatment is 20 kHz to 20 MHz.

[15] The method for promoting an anti-inflammatory effect according to any of

[12] to

[14] , wherein the ultrasonic treatment is performed on cells in a suspended state.

[16] A culture supernatant of inflammation-suppressing cells prepared by the preparation method according to any of [1] to [9].

[17] Extracellular vesicles (exosomes) derived from the culture supernatant according to

[16] .

[18] A method for treating an inflammatory disease, comprising administering to a subject an effective amount of at least one selected from the group consisting of inflammation-suppressing cells prepared by the preparation method according to any of [1] to [9], the culture supernatant according to

[16] , and the extracellular vesicles (exosomes) according to

[17] .

[0009] According to the present invention, the anti-inflammatory effect can be enhanced by treating cells with ultrasound. The mesenchymal stem cells with excellent anti-inflammatory effect obtained by the present invention can act on immune cells in the body to suppress the production of inflammatory factors and promote the production of anti-inflammatory factors, thereby achieving a significant anti-inflammatory effect. The anti-inflammatory cells obtained by the present invention are expected to have excellent therapeutic and ameliorative effects against various inflammatory symptoms and diseases.

[0010] FIG. 1 shows the effect of ultrasonic treatment on the gene expression of various anti-inflammatory cytokines in mesenchymal stem cells. FIG. 2 shows the effect of ultrasonic treatment on the gene expression related to cellular response to various IL-1 (Cellular response to interleukin-1) in mesenchymal stem cells. FIG. 3 shows the effect of ultrasonic treatment on the gene expression related to cellular response to various TNFα in mesenchymal stem cells. FIG. 4 shows the effect of ultrasonic treatment on the gene expression related to cellular response to various LPS in mesenchymal stem cells. FIG. 5 is a schematic diagram showing the method of a co-culture test of ultrasonically treated ADMSCs and THP-1. FIG. 6 shows gene expression of inflammatory factors (TNFα, IL1β) in THP-1 cells after co-culture with sonicated ADMSCs. FIG. 7 shows gene expression of inflammatory factors (IL8) in THP-1 cells after co-culture with sonicated ADMSCs. FIG. 8 shows gene expression of anti-inflammatory factors (IL10, TGFβ) in THP-1 cells after co-culture with sonicated ADMSCs. FIG. 9 shows the amount of TNFα and IL10 produced by THP-1 cells after co-culture with sonicated ADMSCs. FIG. 10 shows the effect of sonication on gene expression of IDO1 and IL4 in mesenchymal stem cells. FIG. 11 shows the effect of sonication on gene expression of HGF in mesenchymal stem cells. FIG. 12 shows the effect of sonication on gene expression of STAR, SRPINE1, PDCD4, HDAC5, LITAF, and ICAM1 in mesenchymal stem cells. Figure 13 is a diagram showing the effect of ultrasonic treatment on the gene expression of ANKRD1, IL24, ACOD1, CD14, and PLSCR4 in mesenchymal stem cells. Figure 14 is a diagram showing the effect of ultrasonic treatment on the gene expression of ABCA1, ADAM9, and BMP6 in mesenchymal stem cells. Figure 15 is a diagram showing the effect of ultrasonic treatment on IL10 and HGF production from mesenchymal stem cells. Figure 16 is a diagram showing the therapeutic effect of hepatitis by administering ultrasonicated ADMSCs to autoimmune hepatitis model mice.

[0011] The method for preparing inflammation-suppressing cells, inflammation-suppressing cells, anti-inflammatory composition, and method for promoting inflammation-suppressing activity of the present invention will be described in detail below.

[0012] <Method for preparing anti-inflammatory cells> The method for preparing anti-inflammatory cells of the present invention is characterized by subjecting cells to ultrasonic treatment. According to the present invention, ultrasonic treatment of cells can significantly enhance the anti-inflammatory effect. Cells such as mesenchymal stem cells with excellent anti-inflammatory effects obtained by the present invention can act on immune cells in the body to suppress the production of inflammatory factors and promote the production of anti-inflammatory factors, and can exert a significant anti-inflammatory effect. The anti-inflammatory cells obtained by the present invention are expected to have excellent therapeutic and ameliorative effects against various inflammatory symptoms and diseases. The method for preparing anti-inflammatory cells of the present invention will be specifically described below.

[0013] [Cells] The cells of the present invention are not particularly limited as long as they are effective in treating diseases, and examples thereof include mesenchymal stem cells, placenta-derived stem cells, umbilical cord blood-derived cells, peripheral blood mononuclear cells (including neutrophils, eosinophils, basophils, lymphocytes, monocytes, etc.), erythrocytes, T cells, NK cells, NKT cells, NKM cells, LAK cells, dendritic cells, fibroblasts, hematopoietic stem cells, iPS cells, ES cells, bone marrow cells, cardiomyocytes, hepatocytes, nerve cells, skin cells, adipocytes, and other cells constituting various tissues. Among these, mesenchymal stem cells, placenta-derived stem cells, umbilical cord blood-derived cells, peripheral blood mononuclear cells, and bone marrow cells are preferred, with mesenchymal stem cells being more preferred, from the viewpoint of excellent effectiveness in treating diseases. Furthermore, the cells of the present invention may be transformants obtained by artificially introducing a specific gene into the above-mentioned cells.

[0014] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (e.g., bone cells, cardiomyocytes, chondrocytes, tenocytes, adipocytes, etc.) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as stromal cells and does not particularly distinguish between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. For example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue and may also be referred to as adipose tissue-derived stromal cells. Of these, from the viewpoints of efficacy in treating diseases, ease of availability, etc., adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and adipose-derived mesenchymal stem cells are most preferred.

[0015] The mesenchymal stem cells of the present invention may be cells provided by, for example, PromoCell, Inc., Lonza, Inc., Biological Industries, Inc., Veritas, Inc., R&D Systems, Inc., Corning, Inc., or cells prepared by methods well known to those skilled in the art. Furthermore, the mesenchymal stem cells may be primary cells isolated from donor tissue or established cell lines.

[0016] As a preparation method well known to those skilled in the art, for example, mesenchymal stem cells can be prepared by obtaining cells from each tissue by enzymatic digestion or physical cutting, then settling the cells, resuspending the cells in an appropriate medium, and culturing them. Furthermore, methods for culturing mesenchymal stem cells include, but are not limited to, suspension culture methods, such as culturing cells on a solid surface such as a petri dish or flask, agitating and culturing spherical cell masses by aggregating the cells, and culturing cells attached to microcarriers by agitation.

[0017] Examples of species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats.

[0018] In the present invention, mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, refer to any cell population containing mesenchymal stem cells derived from various tissues, such as adipose tissue, umbilical cord, and bone marrow, respectively, in which at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% of the cell population are mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0019] In the present invention, the medium used for culturing mesenchymal stem cells is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium may be prepared by adding serum to a basal medium and / or one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. These media may further contain substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc., as needed.

[0020] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, MEM-α medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0021] Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, etc. When serum is used, it may be added to the basal medium at 5 v / v % to 15 v / v %, preferably 10 v / v %.

[0022] In the present invention, the medium used for culturing mesenchymal stem cells is preferably a serum-free medium or a xeno-free medium that does not contain xenogeneic components such as serum. Examples of such media include Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use, manufactured by PromoCell), Mesenchymal Stem Cell Growth Medium XF (Ready-to-use, manufactured by PromoCell), MSCGM Bullet Kit, MSCGM™ Mesenchymal Stem Cell Growth Medium Bullet Kit (manufactured by Lonza), and Xeno-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem (registered trademark) XF, Biological Examples of such a medium include media provided as pre-prepared media for mesenchymal stem cells (stromal cells), such as MesenCult-ACF Plus (manufactured by Veritas), StemXVivotm Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), serum-free medium for adipose-derived stem cells (KBM ADSC-4, manufactured by Kohjin Bio), and serum-free medium for mesenchymal stem cells (R:STEM Medium for hMSC High Growth, manufactured by Rohto).

[0023] The mesenchymal stem cells of the present invention may be cells that have been repeatedly cryopreserved and thawed as appropriate. In the present invention, cryopreservation can be performed by suspending mesenchymal stem cells in a cryopreservation solution well known to those skilled in the art and cooling them. The thawed mesenchymal stem cells may be cultured as appropriate until the next cryopreservation. The mesenchymal stem cells are cultured using the medium described above, and the method is not particularly limited.

[0024] The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following properties: (1) exhibiting adhesiveness to plastic when cultured in a standard medium; (2) being positive for surface antigens CD44, CD73, and CD90, and negative for CD31, CD34, and CD45; and (3) being capable of differentiating into bone cells, adipocytes, and chondrocytes under culture conditions.

[0025] The mesenchymal stem cells of the present invention can be used for various diseases, and specific diseases include GVHD, cartilage degradation, rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout, psoriasis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic valve stenosis, renal failure, nephrotic syndrome, uremia, lupus, pancreatitis, allergy, fibrosis, anemia, atherosclerosis, restenosis, Chemotherapy / radiation-related complications, type I diabetes, type II diabetes, liver failure, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease, nonspecific colitis, intestinal lymphangiectasia, protein-losing enteropathy, Crohn's disease, allergic conjunctivitis, diabetic retinopathy, Sjogren's syndrome, atopic disease, uveitis, allergic rhinitis, food allergy, anaphylaxis, autoimmune disease, drug hypersensitivity, mastocytosis, asthma, asbestosis, silicosis Lung, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, histiocytosis, sarcoidosis, glomerulonephritis, vasculitis, dermatitis, HIV-associated cachexia, cerebral malaria, ankylosing spondylitis, leprosy, COPD, pulmonary fibrosis, fibromyalgia, cancers such as esophageal cancer, gastroesophageal reflux disease, Barrett's esophagus, aplastic anemia, graft-versus-host disease, sickle cell disease, CVID, hyper-IgM syndrome, IgA deficiency, transient hypogammaglobulinemia, X-linked agammaglobulinemia, chronic mucocutaneous candidiasis , DiGeorge syndrome, X-linked lymphoproliferative syndrome, ataxia-telangiectasia, cartilage-hair hypoplasia, combined immunodeficiency, hyper-IgE syndrome, MHC deficiency, severe combined immunodeficiency, Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, IFN-γ receptor deficiency, interleukin (IL)-12 deficiency, IL-12 receptor β1 deficiency, ZAP-70 deficiency, angioedema, and the like.

[0026] The placenta-derived stem cells of the present invention are fetal-derived, low-immunogenic, and tri-germ-differentiating cells, including, but not limited to, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, placental pluripotent cells, and embryonic-like stem cells.

[0027] In the present invention, the cord blood-derived cells are cells contained in cord blood, including hematopoietic stem cells, monocytes, B cells, T cells, NK cells, platelets, and the like.

[0028] Peripheral blood mononuclear cells in the present invention are mononuclear cells isolated from peripheral blood, and include B cells, T cells, NK cells, monocytes, dendritic cells, and the like.

[0029] Bone marrow cells in the present invention include hematopoietic stem cells and mesenchymal stem cells.

[0030] [Ultrasonic Treatment Step] In this step, cells are subjected to ultrasonic treatment. In the present invention, ultrasonic waves refer to sound waves with a frequency that cannot be heard by humans, i.e., high-frequency sound waves of 20 kHz (kilohertz) or higher. In the present invention, there are no particular limitations on the ultrasonic waves, but the frequency is, for example, 20 kHz (kilohertz) to 20 MHz (megahertz), preferably 20 kHz to 10 MHz, more preferably 20 kHz to 5 MHz, even more preferably 20 kHz to 3 MHz, particularly preferably 30 kHz to 2 MHz, even more particularly preferably 40 kHz to 2.0 MHz, and most preferably 500 kHz to 1.5 MHz.

[0031] In the present invention, the intensity of the ultrasonic waves is 50 mW / cm ２ ~5000mW / cm ２ and preferably 100 mW / cm ２ ~4000mW / cm ２ , more preferably 300 mW / cm ２ ~3500mW / cm ２ , and more preferably 500 mW / cm ２ ~3000mW / cm ２ , and even more preferably 1000 mW / cm ２ ~2500mW / cm ２ (Others, for example, 1000 mW / cm ２ ~3000mW / cm ２ , 500 mW / cm ２ ~2500mW / cm ２ Examples include:

[0032] In the present invention, the ultrasonic irradiation time rate (duty cycle) is not particularly limited, but is, for example, 1% to 100%, preferably 10% to 100%, more preferably 50% to 100%, and even more preferably 80% to 100%.

[0033] In the present invention, the ultrasonic treatment time is appropriately adjusted according to the frequency, intensity, irradiation time rate, etc., and is, for example, 10 seconds to 1 hour, preferably 30 seconds to 30 minutes, more preferably 1 minute to 20 minutes, even more preferably 1.5 minutes to 18 minutes, particularly preferably 2 minutes to 15 minutes, and even more particularly preferably 5 minutes to 10 minutes (other examples include 5 minutes to 15 minutes and 2 minutes to 10 minutes).

[0034] In the present invention, when the cells are mesenchymal stem cells, the ultrasonic treatment conditions are a frequency of 20 kHz to 3 MHz and an intensity of 50 mW / cm. ２ ~5000mW / cm ２ , irradiation time rate is 1% to 100%, treatment time is 1 to 20 minutes, frequency is 30 kHz to 2 MHz, intensity is 100 mW / cm ２ ~4000mW / cm ２ , irradiation time rate of 10% to 100%, treatment time of 1.5 minutes to 15 minutes are more preferable, frequency is 40 kHz to 2.0 MHz, intensity is 500 mW / cm ２ ~3000mW / cm ２ More preferably, the irradiation time ratio is 50% to 100%, the treatment time is 2 to 10 minutes, the frequency is 500 kHz to 1.5 MHz, and the intensity is 1000 mW / cm ２ ~3000mW / cm ２ It is particularly preferable that the irradiation time ratio is 80% to 100% and the treatment time is 5 to 10 minutes.

[0035] The ultrasonic generator that can be used in the present invention is not particularly limited as long as it can generate ultrasonic waves, and the principle of ultrasonic generation is also not particularly limited.

[0036] When sonicating cells in the present invention, the cells may be treated by placing a container such as a tube containing a cell suspension in the ultrasonic device, or by treating cells cultured in a culture vessel such as a flask or dish via the culture vessel. Alternatively, an ultrasonic source may be inserted into the culture vessel or cell suspension, and ultrasonic waves may be irradiated directly. In other words, the ultrasonic treatment of cells in the present invention may be indirect irradiation via a tube, culture vessel, or the like, or direct irradiation of the cells themselves or the cell suspension. In the present invention, from the viewpoint of the effects of the present invention and the suitability for use in clinical settings, it is preferable to treat cells by placing a container such as a tube containing a cell suspension in which the cells are suspended in the ultrasonic device. In other words, it is preferable that the cells are suspended when sonicating in the ultrasonic treatment step.

[0037] The tube or the like containing the cell suspension may be prepared by thawing the frozen tube containing the cells in a thermostatic bath or the like, or by transferring the frozen tube to another tube, or by transferring the frozen tube to another tube to which physiological saline, buffer solution, Ringer's solution, or the like has been added. Alternatively, the frozen cells may be transferred to another tube, washed with physiological saline, buffer solution, Ringer's solution, or the like, and then suspended in physiological saline, buffer solution, Ringer's solution, or the like. Furthermore, cultured cells may be collected in a tube or the like, washed, or suspended in physiological saline, buffer solution, Ringer's solution, or the like. The shape, size, etc. of the container, such as a tube, are not limited here.

[0038] The ultrasonic treatment step can be carried out once or multiple times. The number of times of the ultrasonic treatment step is once or twice or more, and specifically, it is preferably 1 to 100 times, more preferably 1 to 10 times, and even more preferably 1 to 5 times.

[0039] The ultrasonic treatment step may be followed by a step of culturing cells. However, it is preferable not to have a step of culturing cells after at least the final ultrasonic treatment step among the ultrasonic treatment steps, and it is more preferable not to have a step of culturing cells after all ultrasonic treatments. It is even more preferable that the final ultrasonic treatment step is performed immediately before administering the anti-inflammatory cells to a subject. Here, "immediately before administering the anti-inflammatory cells to a subject" refers to a period of 20 hours to immediately before administration, preferably 10 hours to immediately before administration, more preferably 5 hours to immediately before administration, even more preferably 2 hours to immediately before administration, and particularly preferably 1 hour to immediately before administration.

[0040] An embodiment of the method for preparing cells for inflammation suppression of the present invention will be specifically described.

[0041] The ultrasonic treatment step in the present invention can be carried out as follows. That is, cryopreserved cells can be thawed by placing the frozen tube in an incubator or the like, and then either the tube or the cells can be transferred to another tube and placed directly in an ultrasonic generator for ultrasonic treatment. Alternatively, cryopreserved cells can be thawed by placing the frozen tube in an incubator or the like, and then the frozen tube can be added with physiological saline, buffer solution, Ringer's solution, etc., or the cells can be transferred to another tube and added with physiological saline, buffer solution, Ringer's solution, etc., and then the tube can be placed in an ultrasonic generator for ultrasonic treatment. Furthermore, cultured cells can be collected in a tube or the like, washed, etc., suspended in physiological saline, buffer solution, Ringer's solution, etc., and then the tube can be placed in an ultrasonic generator for ultrasonic treatment.

[0042] In the ultrasonic treatment, the frequency, intensity, irradiation time rate (duty cycle), and treatment time can be appropriately adjusted depending on the cell type, cell number, purpose, etc., but are performed within the above-mentioned condition ranges. Note that the state of the cells to be ultrasonically treated is preferably a suspended state in a cell suspension.

[0043] The ultrasonic treatment step may be performed only once or may be repeated multiple times. After the ultrasonic treatment step, the cells may be cultured for a certain period of time, and then subjected to another ultrasonic treatment step before being administered to a subject. After the ultrasonic treatment step or the culture step, the cells may be cryopreserved again, and then thawed again before being administered to a subject, or after the second thawing, the cells may be subjected to an ultrasonic treatment step before being administered to a subject.

[0044] The cells may be administered to a subject immediately after sonication or after a lapse of several minutes to 20 hours. The cells after sonication are stored on ice or in a refrigerator to maintain their function.

[0045] The mesenchymal stem cells and other cells with excellent anti-inflammatory effects obtained by the preparation method of the present invention described above can act on immune cells in the body to suppress the production of inflammatory factors, promote the production of anti-inflammatory factors, etc., and as a result, can exert a remarkable anti-inflammatory effect. The anti-inflammatory cells obtained by the present invention are expected to have excellent therapeutic and ameliorative effects against various inflammatory symptoms and diseases.

[0046] It is known that mesenchymal stem cells may not fully exhibit the effects they inherently possess, such as anti-inflammatory effects, depending on conditions such as the donor and the number of passages. However, by using the preparation method of the present invention, the anti-inflammatory effects and other effects can be enhanced even for such cells.

[0047] Inflammatory diseases to which the inflammation-suppressing cells obtained by the preparation method of the present invention can be applied include, for example, allergic inflammatory diseases such as hay fever, delayed-type allergy, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis; neuroinflammatory diseases such as diabetic neuropathy, dementia (Alzheimer's type, etc.), and multiple sclerosis; 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; hepatitis (alcoholic hepatitis, non-alcoholic hepatitis, etc.), asthma, systemic lupus erythematosus, Crohn's disease, atopic dermatitis, arthritis (chronic rheumatoid arthritis, etc.), diabetic neuropathy, bedsores, diabetic skin ulcers, and dementia. inflammatory diseases in which macrophages are involved, such as diabetes; other inflammatory diseases such as arteriosclerosis, gastric ulcer, nephritis (glomerulonephritis, IgA nephropathy, diabetic nephropathy, etc.), periodontal disease (gingivitis, peridontitis, etc.), liver cirrhosis, bronchitis, cerebral infarction, aneurysm, endometriosis, acute respiratory distress syndrome, injuries due to kidney transplantation, 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.

[0048] <Method for Promoting the Inflammatory Cellular Activity> The present invention also includes a method for promoting the inflammatory cell activity, which comprises subjecting cells to ultrasonic treatment. According to the present invention, ultrasonic treatment of cells under specific conditions can promote the anti-inflammatory activity of the cells, and the resulting cells can suppress the production of inflammatory factors from immunocompetent cells and promote the production of anti-inflammatory factors. Therefore, the cells obtained by the method of the present invention can be suitably used for the treatment and amelioration of inflammatory diseases and inflammatory symptoms, as pharmaceuticals, quasi-drugs, cosmetics, and other purposes. Note that the specific content of the method for promoting the inflammatory cell activity of the present invention is the same as that of the method for preparing the inflammatory cell activity of the present invention described above, and therefore the specific description in the section on the method for preparing the inflammatory cell activity of the present invention can be applied.

[0049] <Inflammation-suppressing cells> The present invention also includes inflammation-suppressing cells obtained by the above-mentioned method for preparing inflammation-suppressing cells of the present invention. The inflammation-suppressing cells of the present invention are cells treated with ultrasound by the preparation method of the present invention, and the type of cell and ultrasound conditions (frequency, intensity, exposure time rate, treatment time, etc.) are as described in the section on the preparation method of inflammation-suppressing cells. The cells obtained by the preparation method of the present invention are suitable for use as a pharmaceutical for treating inflammatory diseases.

[0050] When the inflammation-suppressing cells of the present invention are administered to a subject having an inflammatory symptom or an inflammatory disease, the number of cells to be administered, the number of administrations, the administration method, etc. can be appropriately set depending on the condition of the subject, the severity of the symptom, etc.

[0051] The dose (administration amount) of the inflammation-suppressing cells of the present invention may vary depending on the condition of the target patient (body weight, age, symptoms, physical condition, etc.), but from the viewpoint of achieving a sufficient therapeutic effect, a larger amount is preferable, while from the viewpoint of suppressing side effects, a smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 5 x 10 ２ ~1x10 １２ pieces / time, preferably 1 x 10 ４ ~1x10 １１ pieces / time, more preferably 1 x 10 ５ ~1x10 １０ This dose may be administered multiple times as a single dose, or may be administered in divided doses. Generally, when administered to an adult, the number of cells per body weight is 1x10 to 5x10 １０ pieces / kg, preferably 1 x 10 ２ ~5x10 ９ pieces / kg, more preferably 1 x 10 ３ ~5x10 ８ This dose may be administered as a single dose multiple times, or may be administered in divided doses multiple times.

[0052] Furthermore, when cells obtained by the method of the present invention are subjected to ultrasonic treatment under specific conditions, they exhibit enhanced inflammatory response suppression compared to normal cells, and can act on immune cells to promote the production of anti-inflammatory factors or suppress the production of inflammatory factors. Cells obtained by the method of the present invention can also be defined as cells that express, for example, IL4, IL10, HGF, STAR, PDCD4, HDAC5, LITAF, ICAM1, IL24, CD14, PLSCR4, ABCA1, ADAM9, and BMP6 at high levels, or cells that express IDO1, SRPINE1, ANKRD1, and ACOD1 at low levels, compared to cells without ultrasonic treatment. In particular, cells can be defined as cells in which the gene expression levels of IL10, STAR, CD14, and BMP6 are more than double those without ultrasonic treatment, or cells in which the gene expression levels of IDO1, ANKRD1, and ACOD1 are half or less those without ultrasonic treatment. It is unclear what changes occurred in the cells due to ultrasonic treatment to obtain such cells. At least, what has been made clear by the present invention is that cells treated with ultrasonic treatment under the above-described conditions exhibit enhanced inflammatory response suppression compared to normal cells, and can act on immune cells to promote the production of anti-inflammatory factors and suppress the production of inflammatory factors, making them suitable for use as anti-inflammatory cells. Ultrasonic treatment is thought to have many effects on cells. The enhanced anti-inflammatory effect is likely due to a combination of various characteristic changes, including promotion of protein synthesis or inhibition of protein synthesis, inactivation or activation of existing proteins, effects on the cell membrane, and other changes in characteristics. It is thought that it is impossible or impractical to identify all of these characteristic changes through further research, measurement, etc. Therefore, the cells of the present invention are identified by the method as cells obtained by the method for preparing anti-inflammatory cells of the present invention.

[0053] <Cell culture supernatant, extracellular vesicles> The present invention also includes a cell culture supernatant obtained by the method of the present invention. Specifically, for example, it is a cell culture supernatant that has been sonicated under the conditions described above. The inflammation-suppressing cell culture supernatant of the present invention is suitably used for the purpose of treating or ameliorating inflammatory diseases or inflammatory symptoms, as a pharmaceutical product, a quasi-drug, or a cosmetic product, or for other purposes.

[0054] The present invention also includes the cell culture supernatant obtained by the method of the present invention described above, and extracellular vesicles (exosomes) derived from the culture supernatant. Specifically, for example, the cell culture supernatant and extracellular vesicles (exosomes) derived from the culture supernatant are sonicated under the conditions described above. The inflammation-suppressing cell culture supernatant and the extracellular vesicles (exosomes) derived from the culture supernatant of the present invention are suitable for use in treating or ameliorating inflammatory diseases and inflammatory symptoms (inflammation suppression), as pharmaceuticals, quasi-drugs, cosmetics, and other purposes.

[0055] The culture supernatant of the inflammation-suppressing cells obtained by the method of the present invention is not particularly limited as long as it is a supernatant obtained by culturing the inflammation-suppressing cells described above. For example, the inflammation-suppressing cells are typically cultured at a temperature of 30°C to 37°C, in a 2% to 7% CO ２ Under the environment, 5% to 21% O ２ The cells are cultured in an appropriate medium at an appropriate cell density under an appropriate environment. The culture is preferably carried out using a serum-free medium throughout the entire cell culture period. The serum-free medium used here can be the same as that described above in the section "Method for preparing cells for inflammation suppression."

[0056] The timing of collection of the culture supernatant is usually 1 to 5 days, preferably 1 to 4 days, more preferably 1 to 3 days, and even more preferably 2 to 3 days after passage an appropriate number of times taking into consideration the state of the inflammation-suppressing cells. The culture supernatant may be collected only once, or may be collected multiple times over multiple days.

[0057] The term "culture supernatant" as used herein refers to a culture medium (post-culture culture medium) obtained by culturing the anti-inflammatory cells in a culture medium in which the anti-inflammatory cells can grow or survive under conditions that allow the cells to grow or survive, from which the anti-inflammatory cells and other substances have been removed. However, for convenience, the term "culture supernatant for anti-inflammatory cells" as used herein also refers to a culture medium from which at least a portion of components that do not contribute to the effects of the present invention have been further removed, such as residual medium components (components of the culture medium before culture that remain in the culture medium after culture) and water content of the culture medium. From the standpoint of simplicity, it is preferable to use the culture medium from which the anti-inflammatory cells have been removed as is as the culture supernatant.

[0058] Extracellular vesicles (exosomes) derived from the culture supernatant of inflammation-suppressing cells obtained by the method of the present invention can be obtained by conventionally known methods, such as separating the culture supernatant of inflammation-suppressing cells obtained by the above-mentioned method using ultracentrifugation, density gradient centrifugation, affinity purification, size exclusion chromatography, various exosome isolation kits, etc. Preferred methods for separating exosomes are ultracentrifugation, affinity purification, and size exclusion chromatography.

[0059] <Anti-inflammatory Composition> The present invention also encompasses an anti-inflammatory composition containing at least one selected from the group consisting of the above-described inflammation-suppressing cells of the present invention, their culture supernatant, and extracellular vesicles (exosomes) derived from the culture supernatant. As described above, the inflammation-suppressing cells of the present invention, their culture supernatant, and extracellular vesicles (exosomes) derived from the culture supernatant can suppress the production of inflammatory factors from immunocompetent cells and promote the production of anti-inflammatory factors. Therefore, the anti-inflammatory composition of the present invention can be suitably used for the treatment and amelioration of inflammatory diseases and inflammatory symptoms. The inflammation-suppressing cells of the present invention are cells treated with ultrasound of a specific intensity using the method for preparing inflammation-suppressing cells of the present invention. The cell type and ultrasound conditions (frequency, intensity, exposure time rate, treatment time, etc.) are as described in the section on the method for preparing inflammation-suppressing cells.

[0060] The anti-inflammatory composition of the present invention may contain, in addition to the above-mentioned inflammation-suppressing cells, other cells as long as the effects of the present invention are not impaired, and may contain pharmaceutically acceptable carriers and additives according to standard methods depending on the intended use and form. Examples of such carriers and additives include, but are not limited to, isotonicity agents, thickeners, sugars, sugar alcohols, preservatives, bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, and colorants.

[0061] When the anti-inflammatory composition of the present invention is administered to a subject, its dose (administration amount) may vary depending on the condition of the subject patient (body weight, age, symptoms, physical condition, etc.), but from the viewpoint of achieving a sufficient therapeutic effect, a larger dose (administration amount) of the inflammation-suppressing cells contained therein is preferable, while from the viewpoint of suppressing side effects, a smaller dose tends to be preferable. Usually, when administered to an adult, the number of inflammation-suppressing cells is 5 x 10 ２ ~1x10 １２ pieces / time, preferably 1 x 10 ４ ~1x10 １１ pieces / time, more preferably 1 x 10 ５ ~1x10 １０ This dose may be administered multiple times as a single dose, or may be administered in divided doses. Generally, when administered to an adult, the number of cells per body weight is 1x10 to 5x10 １０ pieces / kg, preferably 1 x 10 ２ ~5x10 ９ pieces / kg, more preferably 1 x 10 ３ ~5x10 ８The dose is 0.001 pg / mL to 10 mg / mL, preferably 0.005 pg / mL to 1 mg / mL. When the anti-inflammatory composition of the present invention contains extracellular vesicles (exosomes), the amount of exosome-derived protein contained in the anti-inflammatory composition is 0.001 pg / mL to 10 mg / mL, preferably 0.005 pg / mL to 1 mg / mL. When administered to an adult, the dose is 0.001 pg / dose to 10 mg / dose, preferably 0.005 pg / dose to 1 mg / dose, in terms of the amount of exosome-derived protein.

[0062] The present invention also includes inventions of anti-inflammatory agents, anti-inflammatory pharmaceutical compositions, and therapeutic agents for inflammatory diseases, which contain the inflammation-suppressing cells of the present invention described above, and which are substantially the same as the anti-inflammatory compositions described above.

[0063] The present invention also includes a method for treating inflammatory diseases, which comprises administering to a subject an effective amount of at least one selected from the group consisting of the inflammation-suppressing cells of the present invention, their culture supernatant, and extracellular vesicles (exosomes) derived from the culture supernatant. The descriptions in the respective sections apply to the inflammation-suppressing cells of the present invention, their culture supernatant, and extracellular vesicles (exosomes) derived from the culture supernatant.

[0064] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0065] 1. Effect of ultrasonic treatment on gene expression in adipose-derived mesenchymal stem cells (1) Cryopreserved human adipose-derived mesenchymal stem cells (Lonza, Cat: 22TL108471, hereinafter also referred to as "ADMSC") were removed from liquid nitrogen and thawed in a 37°C water bath for 2 minutes. Then, they were immersed in another water bath connected to an ultrasonic generator (Opharmic) and subjected to ultrasonic treatment. The ultrasonic frequency was 1.5 megahertz (MHz), and the intensity was 30 mW / cm. ２ , 670 mW / cm ２ , 2500mW / cm ２The irradiation time rate (duty cycle) was 100%, and the treatment time was 10 minutes. The control group was not subjected to ultrasonic treatment (CNTL). After ultrasonic treatment, the cells were sonicated at 35 mm ２ 2 mL of RIM medium (Rohto Pharmaceutical Co., Ltd.) was used to culture the cells at 5,000 cells / cm ２ The cells were cultured at a density of 1000 kJ / ml and incubated overnight in a humidified incubator at 37°C. The next day, the medium was aspirated, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were then lysed in lysis buffer to prepare a cell lysate. RNA samples were extracted from the cell lysate according to the manufacturer's protocol (NucleoSpin RNA Plus (MACHEREY-NAGEL, Cat. No. 740984.250)). RNA concentrations were measured using a Nanodrop spectrophotometer (Thermo Scientific), and each sample was subjected to RNA sequencing (RNASeq) (Novogene). The specific measurement results are as follows:

[0066] The expression intensities of genes involved in anti-inflammatory cytokines (Figure 1), cellular response to IL-1 (Cellular response to interleukin-1: Figure 2), cellular response to TNFα (Cellular response to TNFα: Figure 3), and cellular response to LPS (Cellular response to LPS: Figure 4) were measured, and the analysis results are shown in each figure. These figures are heat maps created based on the above RNASeq data using Novogene's Novomagic platform. In each figure, A1: Untreated (CNTL), A2: 30 mW / cm ２ , A3: 670mW / cm ２ , A4: 2500mW / cm ２ In addition, in the heat map, areas that are originally red are marked with a "+" and areas that are originally blue are marked with a "-" so that increases or decreases in gene expression can be distinguished even when the figure is in black and white.

[0067] As shown in each figure, it was found that ultrasonic treatment of human adipose-derived mesenchymal stem cells strongly tends to suppress the gene expression of factors involved in promoting inflammation and enhance the gene expression of factors involved in suppressing inflammation.

[0068] 2. Co-culture test of sonicated ADMSCs and immune cells (qPCR analysis) Figure 5 shows a schematic diagram of the test method. Specifically, the test was performed using cell culture inserts (pore size 0.4 μm; Corning, NY, USA). First, human monocyte cell line THP-1 (3 × 10 ４ pieces / cm ２ ) were seeded in the lower chamber of a transwell and treated with 200 ng / mL PMA in 10% FBS-supplemented RPMI 1640 for 72 hours to stimulate differentiation. Next, the cells were washed three times with phosphate-buffered saline (PBS), and 1 mL of RPMI medium supplemented with 1 μg / mL LPS (RPMI-LPS) was added. MSC cells (8 x 10) sonicated under each condition were cultured in a similar manner to "1. Effect of sonication on adipose-derived mesenchymal stem cells on gene expression (1)." ３ pieces / cm ２ ) were seeded onto the inserts and cultured for 6 hours to allow cells to adhere. Then, the inserts containing sonicated MSCs were replaced with RPMI-LPS and placed directly on top of the 12-well plate containing THP-1 cells. They were cultured in this state for an additional 48 hours.

[0069] After 48 hours of incubation, THP-1 cells were harvested and washed twice with PBS. Then, they were lysed in LBP lysis buffer according to the manufacturer's protocol. RNA was collected using NucleoSpin® RNA plus (TAKARA, product number: 740984.250). Reverse transcription was performed using Primescript RT Master Mix (TAKARA) according to the manufacturer's protocol. Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (TAKARA) on a QuantStudio 1 Real-Time PCR System. Technical triplicates were run for each cDNA sample using the fast program. The results are shown in Figures 6-8.

[0070] As shown in Figures 6 and 7, the gene expression of inflammatory factors (TNFα, IL1β, and IL8) in THP-1 cells was suppressed by co-culture with ultrasonicated ADMSCs compared to that of untreated ADMSCs. The effect was observed for TNFα and IL1β when the ultrasonic treatment intensity for ADMSCs was 30 mW / cm. ２ In the case of 2500 mW / cm ２ For IL8, the ultrasonic treatment intensity for ADMSCs was 30 mW / cm ２ In the case of ADMSCs treated with ultrasound, gene expression tended to be enhanced rather than that in untreated ADMSCs, but the ultrasound treatment intensity was 2500 mW / cm ２ In addition, as shown in Figure 8, gene expression of anti-inflammatory factors (IL10, TGFβ) in THP-1 cells was promoted by co-culture with ultrasound-treated ADMSCs compared to that in the case of ultrasound-untreated ADMSCs. This effect was observed when the ultrasound treatment intensity for ADMSCs was 30 mW / cm. ２ In the case of 2500 mW / cm ２ As mentioned above, 2500 mW / cm was higher. ２ ADMSCs treated with ultrasound at an intensity of 1000 Hz suppressed the gene expression of inflammatory factors and promoted the gene expression of anti-inflammatory factors in co-cultured THP-1.

[0071] 3. Co-culture test of ultrasonically treated ADMSCs and immune cells (ELISA) Co-culture (48 hours) of ultrasonically treated ADMSCs and immune cells was carried out in the same manner as in "2. Co-culture test of ultrasonically treated ADMSCs and immune cells."

[0072] After 48 hours of co-culture of THP1 and MSC, the medium was centrifuged at 400 × g for 5 minutes at room temperature. It was then filtered through a 0.22 μM filter and stored at -80°C before analysis. TNFα and IL-10 were measured using commercially available ELISA kits from Abeam (references: ab181421 and ab185986). All ELISA kits were performed according to the manufacturer's instructions. The sensitivity of the TNFα and IL-10 assays was 4.32 pg / ml and 1.4 pg / ml, respectively. For each assay, a 50 μL aliquot of each supernatant was assayed in duplicate. The results are shown in Figure 9.

[0073] As shown in Figure 9, the production of TNFα, an inflammatory factor, in THP-1 cells was suppressed by co-culture with ultrasonically treated ADMSCs compared to that in the case of untreated ADMSCs. ２ In the case of ADMSCs treated with ultrasound, the production of TNFα tended to be enhanced compared to that of ADMSCs not treated with ultrasound, but the ultrasound treatment intensity was 2500 mW / cm ２ Furthermore, the production of IL10, an anti-inflammatory factor, was promoted by co-culture with ultrasonically treated ADMSCs compared to that of untreated ADMSCs, and this effect was greatest when the ultrasonic treatment intensity for ADMSCs was 30 mW / cm. ２ In the case of 2500 mW / cm ２ was higher.

[0074] 4. Effect of ultrasonic treatment on gene expression in adipose-derived mesenchymal stem cells (2) Cryopreserved human adipose-derived mesenchymal stem cells (Lonza, Cat: 22TL108471, hereinafter also referred to as "ADMSC") were removed from liquid nitrogen and thawed in a 37°C water bath for 2 minutes. Then, they were immersed in another water bath connected to an ultrasonic generator (Opharmic) and subjected to ultrasonic treatment. The ultrasonic frequency was 1.5 megahertz (MHz), and the intensity was 30 mW / cm. ２ , 670 mW / cm ２ , 2500mW / cm ２The irradiation time rate (duty cycle) was 100%, and the treatment time was 10 minutes. The control group was not subjected to ultrasonic treatment (CNTL). After ultrasonic treatment, the cells were sonicated at 35 mm ２ 2 mL of RIM medium (Rohto Pharmaceutical Co., Ltd.) was used to culture the cells at 5,000 cells / cm ２ The cells were cultured at a density of 1000 μg / ml and incubated overnight in an incubator at 37°C. The next day, the medium was aspirated, and the cells were washed twice with phosphate-buffered saline (PBS). Then, the cells were lysed in a lysis buffer to prepare a cell lysate.

[0075] RNA was collected using NucleoSpin® RNA plus (TAKARA, product number: 740984.250). Reverse transcription was performed using Primescript RT Master Mix (TAKARA) according to the manufacturer's protocol. Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (TAKARA) on a QuantStudio 1 Real-Time PCR System. Technical triplicates were run for each sample using the fast program for cDNA samples. The results are shown in Figures 10-14.

[0076] The expression levels of genes involved in anti-inflammatory cytokines (Figures 10 and 11) and cellular response to LPS (Figures 12 to 14) were measured, and the results are shown in each figure. In each figure, the horizontal axis represents ultrasonic treatment intensity (mW / cm ２ ), and the vertical axis represents the relative intensity of mRNA expression of each factor.

[0077] As shown in each figure, it was found that ultrasonic treatment of human adipose-derived mesenchymal stem cells strongly tends to suppress the gene expression of factors involved in promoting inflammation and enhance the gene expression of factors involved in suppressing inflammation.

[0078] 5. Effect of ultrasonic treatment of adipose-derived mesenchymal stem cells on IL-10 and HGF production Cryopreserved human adipose-derived mesenchymal stem cells (Lonza, Cat: 22TL108471, hereinafter also referred to as "ADMSC") were removed from liquid nitrogen and thawed in a 37°C water bath for 2 minutes. Then, they were immersed in another water bath connected to an ultrasonic generator (Opharmic) and subjected to ultrasonic treatment. The ultrasonic frequency was 1.5 megahertz (MHz), and the intensity was 2500 mW / cm. ２ The irradiation time rate (duty cycle) was 100%, and the treatment time was 10 minutes. The control group was not subjected to ultrasonic treatment (CNTL). After ultrasonic treatment, the cells were sonicated at 35 mm ２ 2 mL of RIM medium (Rohto Pharmaceutical Co., Ltd.) was used to culture the cells at 5,000 cells / cm ２ The cells were cultured at a density of 1000×g and incubated overnight in an incubator at 37° C. The next day, the medium was collected and subjected to ELISA testing.

[0079] IL-10 and HGF were measured using commercially available ELISA kits (ab185986, RK00091) from Abeam and ABclonal. All procedures were performed according to the manufacturer's instructions. The sensitivity of IL-10 and HGF was 1.4 pg / ml and 62.5 pg / ml, respectively. For each assay, 50 μL aliquots of each supernatant were assayed in duplicate. The results are shown in Figure 15.

[0080] As shown in Figure 15, it was found that ultrasonic treatment of human adipose-derived mesenchymal stem cells tends to enhance the production of IL-10 and HGF, which are known to be involved in inflammation suppression.

[0081] 6. Confirmation of therapeutic efficacy using a mouse model of autoimmune hepatitis. Cryopreserved human adipose-derived mesenchymal stem cells (Lonza, Cat: 22TL108471, hereinafter referred to as "ADMSCs") were removed from liquid nitrogen, thawed in a 37°C water bath for 2 minutes, and suspended in DMEM. After centrifugation, the cells suspended in HBSS were transferred to a tube designed for ultrasonication and sonicated in an ultrasonic generator (Opharmic). The ultrasonic frequency was 1.5 megahertz (MHz) and the intensity was 2500 mW / cm2. The exposure time rate (duty cycle) was 100%, and the treatment time was 10 minutes. ADMSCs that had not been sonicated were used as a negative control. Each cell was suspended in HBSS and used for administration.

[0082] Separately, hepatitis was induced in mice (BALB / cJ (Oriental Yeast Co., Ltd.), 9-week-old, female) by a single administration of 15 mg / kg of concanavalin A (Sigma-Aldrich). The above-mentioned ADMSCs that had not been subjected to ultrasonic treatment (CNTL ADMSCs) and ADMSCs that had been irradiated with ultrasonic waves (US ADMSCs) were injected into these hepatitis-induced mice at a concentration of 5 × 10 ７ The cells were administered intravenously in a single dose at 1000kJ / kg. Blood was collected 24 hours after administration of the cells to the mice, and serum AST levels were measured using a DriChem (Fujifilm Medical Co., Ltd.). The results are shown in Figure 16. In the figure, "Normal" indicates serum from normal BALB / cJ mice, "CNTL·ADMSC" indicates serum from hepatitis-induced mice administered with CNTL·ADMSC, and "US·ADMSC" indicates serum from hepatitis-induced mice administered with US·ADMSC.

[0083] The AST suppression effect in serum of hepatitis-induced mice treated with US·ADMSC was calculated from the obtained AST measurement values ​​and compared with the CNTL·ADMSC group using the following formula: AST suppression rate in serum by US·ADMSC administration (%) = [(AST level in CNTL·ADMSC group) - (AST level in US·ADMSC group)] / (AST level in CNTL·ADMSC group) × 100

[0084] As shown in Figure 16, serum AST was suppressed by 61.8% in the ultrasonically treated ADMSC-administered group (US-ADMSC) compared to the non-ultrasonic treated ADMSC-administered group (CNTL-ADMSC). As described above, it was confirmed that ultrasonic treatment of ADMSCs enhanced the anti-inflammatory effect of ADMSCs, resulting in a more significant reduction in liver damage.

[0085] According to the present invention, the anti-inflammatory effect can be significantly enhanced by treating cells with ultrasound. The mesenchymal stem cells with excellent anti-inflammatory effect obtained by the present invention can act on immune cells in the body to suppress the production of inflammatory factors and promote the production of anti-inflammatory factors, thereby achieving a significant anti-inflammatory effect. The anti-inflammatory cells obtained by the present invention are expected to have excellent therapeutic and ameliorative effects against various inflammatory symptoms and diseases.

Claims

1. A method for preparing cells for inflammation suppression, which comprises subjecting cells to ultrasonic treatment.

2. The ultrasonic intensity in the ultrasonic treatment is 50 mW / cm ２ ~5000mW / cm ２ 2. The method for preparing inflammation-suppressing cells according to claim 1, wherein 3. A method for preparing inflammation-suppressing cells according to claim 1 or 2, wherein the frequency of the ultrasonic treatment is 20 kHz to 20 MHz.

4. A method for preparing cells for suppressing inflammation described in any one of claims 1 to 3, wherein the cells are mesenchymal stem cells.

5. A method for preparing cells for suppressing inflammation according to claim 4, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

6. A method for preparing inflammation-suppressing cells according to any one of claims 1 to 5, wherein the ultrasonic treatment step is carried out once or multiple times.

7. The method for preparing cells for suppressing inflammation according to claim 6, which does not include a step of culturing the cells after the ultrasonic treatment.

8. The method for preparing inflammation-suppressing cells according to claim 7, wherein the final ultrasonic treatment step is carried out immediately before administering the cells to a subject.

9. A method for preparing cells for inflammation suppression according to any one of claims 1 to 8, wherein the ultrasonic treatment is performed on cells in a suspended state.

10. Cells for suppressing inflammation prepared by the preparation method described in any one of claims 1 to 9.

11. An anti-inflammatory composition comprising the inflammation-suppressing cells described in claim 10.

12. A method for promoting the anti-inflammatory activity of cells, which comprises subjecting the cells to ultrasonic treatment.

13. The ultrasonic intensity in the ultrasonic treatment is 50 mW / cm ２ ~5000mW / cm ２ The method for promoting an anti-inflammatory effect according to claim 12, wherein 14. The method for promoting an anti-inflammatory effect according to claim 12 or 13, wherein the frequency of the ultrasonic treatment is 20 kHz to 20 MHz.

15. A method for promoting an anti-inflammatory effect according to any one of claims 12 to 14, wherein the ultrasonic treatment is performed on cells in a suspended state.

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