Method for preparing cells for disease treatment, method for promoting cell migration ability, method for promoting cell proliferation ability, and cells obtained by these methods

Abstract

An object of the present invention is to provide a method for obtaining cells with high migration ability and high proliferation ability, which are cells with enhanced therapeutic effects against diseases. The present invention relates to a method for preparing cells for disease treatment, a method for promoting the migration ability of cells, and a method for promoting the proliferation ability of cells, which are characterized by subjecting cells to ultrasonic treatment. It is preferable that the frequency in the ultrasonic treatment is 30 kilohertz to 2.0 megahertz, and the cells are mesenchymal stem cells. The present invention also includes cells obtained by the preparation method of the present invention.

Description

[Technical field]

[0001] The present invention relates to a method for preparing cells for the treatment of a disease, a method for promoting the migration ability of cells, a method for promoting the proliferation ability of cells, and cells obtained by these methods. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are multipotent precursor 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 fat, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Non-Patent Documents 1 and 2). Recently, it has been found that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc. Therefore, mesenchymal stem cells are sometimes called stromal cells.

[0003] It is well known that mesenchymal stem cells have tissue repair and immunosuppressive effects and are therefore used in regenerative medicine. However, the mechanism by which mesenchymal stem cells exert their effects has not yet been fully elucidated. Recently, it has been reported that mesenchymal stem cells have the property of actively accumulating in inflammatory tissues and tumor tissues in the body, and it is thought that mesenchymal stem cells secrete cytokines, exosomes, etc. in inflammatory tissues and tumor tissues in the body and promote the repair of inflammatory tissues by acting on surrounding cells (Non-Patent Documents 3 and 4). Under these circumstances, there is a demand for technology that can further enhance the therapeutic effects of mesenchymal stem cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Pittenger FMet al.Science ,(1999),284,pp.143-147 [Non-Patent Document 2] Cell Transplantation, (2016), 25, pp. 829-848 [Non-Patent Document 3] iScience, (2019) 15, pp.421-438 [Non-Patent Document 4] Int J Med Sci. (2018), 15(10),pp.1051-1061 Summary of the Invention [Problem to be solved by the invention]

[0005] In the above-mentioned circumstances, an object of the present invention is to provide a method for obtaining cells with high migration ability and high proliferation ability, which are cells with improved therapeutic effects against diseases. [Means for solving the problem]

[0006] The present inventors believed that increasing the accumulation of cells in inflamed tissues and tumor tissues would lead to a greater therapeutic effect of cells, and conducted intensive research to solve the above problems. As a result, the present inventors discovered that ultrasonic treatment of mesenchymal stem (stromal) cells (MSCs) is effective in increasing the migration and proliferation capabilities of mesenchymal stem cells, and completed the present invention. According to the present invention, cells with high migration ability and high proliferation ability can be provided. That is, the gist of the present invention is as follows. [1] A method for preparing cells for disease treatment, comprising subjecting cells to ultrasonic treatment. [2] The method for preparing cells for the treatment of a disease according to [1], wherein the frequency in the ultrasonic treatment is 30 kilohertz to 2.0 megahertz. [3] The method for preparing cells for disease treatment described in [1] or [2], wherein the cells are mesenchymal stem cells. [4] A method for promoting cell migration, comprising subjecting cells to ultrasonic treatment. [5] The method for promoting cell migration according to [4], wherein the frequency in the ultrasonic treatment is 30 kilohertz to 2.0 megahertz. [6] The method for promoting the migration ability of a cell described in [4] or [5], wherein the cell is a mesenchymal stem cell. [7] A method for promoting cell proliferation, comprising subjecting cells to ultrasonic treatment. [8] The method for promoting cell proliferation according to [7], wherein the frequency in the ultrasonic treatment is 30 kilohertz to 2.0 megahertz. [9] The method for promoting the migration ability of a cell described in [7] or [8], wherein the cell is a mesenchymal stem cell.

[10] Cells treated with ultrasound at frequencies between 30 kilohertz and 2.0 megahertz.

[11] Mesenchymal stem cells treated with ultrasound at frequencies between 30 kHz and 2.0 MHz. Effect of the Invention

[0007] According to the present invention, a novel method for promoting the migration ability of cells and cells with promoted migration ability can be provided. According to the present invention, a novel method for promoting the proliferation ability of cells and cells with promoted proliferation ability can be provided. According to the present invention, since cells with excellent migration ability and cells with excellent proliferation ability can be obtained, it is possible to accumulate a large number of cells at disease sites such as inflamed tissues and tumor tissues, and it is believed that the effect of disease treatment by cells can be improved. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the cell proliferation ability of mesenchymal stem cells. [Diagram 2] FIG. 2 shows the cell proliferation ability of mesenchymal stem cells. [Diagram 3] FIG. 3 shows the cell proliferation ability of mesenchymal stem cells. [Figure 4] FIG. 4 shows the cell proliferation ability of mesenchymal stem cells. [Diagram 5] FIG. 5 shows the cell proliferation ability of mesenchymal stem cells. [Figure 6] FIG. 6 shows the results of the scratch assay. [Figure 7]FIG. 7 quantifies the results of the scratch assay. [Figure 8] FIG. 8 shows stained cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 9] FIG. 9 shows the number of cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 10] FIG. 10 shows stained cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 11] FIG. 11 shows the number of cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 12] FIG. 12 shows the results of repair over time in the scratch assay. [Figure 13] FIG. 13 quantifies the percentage of repair after 9 hours in the scratch assay. [Figure 14] FIG. 14 shows stained cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 15] FIG. 15 shows the number of cells that migrated to the lower surface of the Transwell in the migration assay. [Figure 16] FIG. 16 shows the results of repair over time in the scratch assay. [Figure 17] FIG. 17 quantifies the percentage of repair after 9 hours in the scratch assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The method for preparing cells for treating a disease, the method for promoting the migration ability of cells, the method for promoting the proliferation ability of cells, and the cells obtained by these methods of the present invention will be described in detail below.

[0010] <Method of preparing cells for disease treatment> The method for preparing cells for disease treatment of the present invention is characterized by subjecting cells to ultrasonic treatment. According to the present invention, by subjecting cells to ultrasonic treatment under specific conditions, it is possible to enhance the migration ability of the cells and provide cells that have a high accumulation tendency at disease sites such as inflamed tissues and tumor tissues, thereby further enhancing the disease treatment effect. The method for preparing cells for disease treatment of the present invention is described below.

[0011] [cell] The cells in the present invention are not particularly limited as long as they are effective in treating a disease, and examples thereof include mesenchymal stem cells, placenta-derived stem cells, 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, cardiac myocytes, hepatic cells, nerve cells, skin cells, adipocytes, and other cells constituting various tissues. Among these, mesenchymal stem cells, placenta-derived stem cells, cord blood-derived cells, peripheral blood mononuclear cells, and bone marrow cells are preferred, and mesenchymal stem cells are more preferred, from the viewpoint that the migration ability is further improved by ultrasonic treatment and that they are highly effective in treating a disease.

[0012] 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 (bone cells, cardiac muscle cells, chondrocytes, tendon cells, 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 interstitial 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, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. 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 standpoint of efficacy in treating diseases, ease of availability, and the like, 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.

[0013] The mesenchymal stem cells in the present invention may be, for example, cells provided by PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, etc., or may be cells that can be prepared by methods well known to those skilled in the art. In addition, the mesenchymal stem cells may be primary cells isolated from donor tissue, or may be established cell lines.

[0014] As a preparation method well known to those skilled in the art, for example, cells can be obtained from each tissue by enzymatic digestion or physical cutting, then the cells are precipitated, resuspended in an appropriate medium, and cultured to prepare the mesenchymal stem cells of the present invention. In addition, cell culture methods include, but are not limited to, suspension culture methods such as a method of culturing cells on a solid surface such as a petri dish or flask, a method of agglomerating cells to form spherical cell masses and culturing them with stirring, and a method of adhering cells to a microcarrier and culturing them by stirring.

[0015] In the present invention, species of mesenchymal stem cells include human, horse, cow, sheep, pig, dog, cat, rabbit, mouse, and rat.

[0016] In the present invention, the term "mesenchymal stem cells derived from each tissue, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells" refers to any cell population containing mesenchymal stem cells derived from each tissue, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. 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 each tissue, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0017] The medium used for culturing the cells of the present invention 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 adding 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 necessary.

[0018] 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.

[0019] 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%.

[0020] The mesenchymal stem cells in 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 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 a medium capable of culturing the above-mentioned mesenchymal stem cells, and are not particularly limited.

[0021] The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following characteristics: (1) When cultured in standard medium, it exhibits adhesion to plastic. (2) surface antigens CD44, CD73, and CD90 are positive, and CD31, CD34, and CD45 are negative; and (3) They can be differentiated into bone cells, adipocytes, and chondrocytes under appropriate culture conditions.

[0022] The mesenchymal stem cells of the present invention can be used for various diseases, and specific examples of such 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, allergies, fibrosis, anemia, atherosclerosis, restenosis, Chemotherapy / radiation-related complications, diabetes mellitus type I, diabetes mellitus type II, 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 diseases, uveitis, allergic rhinitis, food allergies, anaphylaxis, autoimmune diseases, drug hypersensitivity, mastocytosis, asthma, asbestosis, silicosis Lungs, 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, esophageal and other cancers, 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-gamma receptor deficiency, interleukin (IL)-12 deficiency, IL-12 receptor β1 deficiency, ZAP-70 deficiency, and angioedema.

[0023] The placenta-derived stem cells of the present invention are cells derived from a fetus, have low immunogenicity, and have the ability to differentiate into three germ layers. Placenta-derived stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, placental pluripotent cells, and embryonic-like stem cells.

[0024] In the present invention, the cord blood-derived cells are cells contained in umbilical cord blood, and include hematopoietic stem cells, monocytes, B cells, T cells, NK cells, platelets, and the like.

[0025] In the present invention, peripheral blood mononuclear cells refer to mononuclear cells separated from peripheral blood, and include B cells, T cells, NK cells, monocytes, dendritic cells, and the like.

[0026] Bone marrow cells in the present invention include hematopoietic stem cells and mesenchymal stem cells.

[0027] [Ultrasound] In the present invention, ultrasonic waves refer to sound waves with a frequency that cannot be heard by humans, and refer to sound waves with a high frequency of 20 kHz (kilohertz) or higher. In the present invention, there is no particular limitation as long as it is 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 1.5 MHz, and most preferably 500 kHz to 1.5 MHz.

[0028] In the present invention, the intensity of the ultrasonic waves is not particularly limited, but is, for example, 0.1 mW / cm 2 ~200mW / cm 2 , preferably 0.6 mW / cm 2 ~100mW / cm 2 , and more preferably 1 mW / cm 2 ~75mW / cm 2 , more preferably 10 mW / cm 2 ~50mW / cm 2 It is.

[0029] In the present invention, the ultrasonic irradiation time rate (duty cycle) is not particularly limited, but is, for example, 1% to 95%, preferably 5% to 75%, more preferably 10% to 60%, and further preferably 20% to 50%.

[0030] 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 15 minutes, and particularly preferably 2 minutes to 10 minutes.

[0031] In the present invention, when the cells are mesenchymal stem cells, the conditions for ultrasonic treatment are a frequency of 20 kHz to 3 MHz and an intensity of 0.6 mW / cm. 2 ~100mW / cm 2 The irradiation time rate is preferably 5% to 75%, the treatment time is preferably 1 to 20 minutes, the frequency is 30 kHz to 2 MHz, and the intensity is 1 mW / cm 2 ~75mW / cm 2 More preferably, the irradiation time rate is 10% to 60%, the treatment time is 1.5 minutes to 15 minutes, the frequency is 40 kHz to 1.5 MHz, and the intensity is 10 mW / cm 2 ~50mW / cm 2 It is more preferable that the irradiation time rate is 20% to 50% and the treatment time is 2 minutes to 10 minutes.

[0032] 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 ultrasonic generation principle is not particularly limited either. An example of the ultrasonic generator is the METRON ACCUSONIC PLUS ULTRASOUND THERAPY UNIT (MODEL AP100, Metron Medical Australia Pty Ltd).

[0033] When ultrasonically treating cells in the present invention, a culture vessel such as a flask or dish or a tube containing a cell suspension may be set in an ultrasonic device for treatment, or an ultrasonic source may be inserted into the culture vessel or cell suspension and ultrasonic waves may be directly irradiated. That is, ultrasonic treatment of cells in the present invention may be indirect irradiation via the culture vessel in which the culture was performed, 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, it is preferable to set the culture vessel in the ultrasonic device for treatment.

[0034] One embodiment of the method for preparing cells for treating a disease of the present invention will be specifically described.

[0035] The cells are seeded in a culture vessel, cultured for 0 to 4 days, preferably 1 to 2 days, and then the culture vessel is placed in an ultrasonic generator in the cultured state, and ultrasonic treatment is performed from the bottom of the culture vessel. The frequency, intensity, exposure time rate (duty cycle), and treatment time can be appropriately adjusted according to the cell type, cell number, purpose, etc., but are performed within the above-mentioned condition range. The state of the cells to be ultrasonically treated may be the state in which they are cultured in the culture vessel as described above, the state in which the cultured cells are collected in a test tube, etc., or the state in which the frozen cells are thawed, etc.

[0036] The cells after ultrasonic treatment may be used for disease treatment as they are, or may be cultured for a certain period of time before being used for disease treatment. Also, the cells after ultrasonic treatment may be cultured and then passaged one or more times before being used for disease treatment.

[0037] The cells obtained by the method for preparing cells for disease treatment of the present invention have higher migration ability and are also excellent in wound repair ability as shown by scratch assay compared to normal cells. Therefore, according to the present invention, it is possible to provide cells that have a high accumulation at disease sites, and therefore it is possible to further improve the disease treatment effect against inflammatory diseases, tumors, etc. In particular, when the cells obtained by the method for preparing cells for disease treatment of the present invention are mesenchymal stem cells, it is possible to further improve the therapeutic effect in the treatment of various diseases for which the above-mentioned mesenchymal stem cells can be used. The cells prepared by the method of the present invention are suitably used as a medicine for disease treatment.

[0038] <Method for promoting cell migration> The present invention also includes a method for promoting cell migration, characterized by subjecting cells to ultrasonic treatment. According to the present invention, by subjecting cells to ultrasonic treatment under specific conditions, it is possible to promote the migration of cells and increase the accumulation of cells at disease sites. Therefore, the cells obtained by the method of the present invention can be suitably used for treating diseases such as inflammatory diseases and tumors. Note that the specific content of the method for promoting cell migration of the present invention is the same as that of the above-mentioned method for preparing cells for disease treatment of the present invention, and therefore the specific explanation can be applied to the explanation in the section on the method for preparing cells for disease treatment.

[0039] <Method for promoting cell proliferation> The present invention also includes a method for promoting cell proliferation, characterized by subjecting cells to ultrasonic treatment. According to the present invention, by subjecting cells to ultrasonic treatment under specific conditions, it is possible to promote the proliferation of cells, and it becomes possible to accumulate a large number of cells at the disease site. Therefore, the cells obtained by the method of the present invention can be suitably used for treating diseases such as inflammatory diseases and tumors. Note that the specific content of the method for promoting cell proliferation of the present invention is the same as that of the above-mentioned method for preparing cells for disease treatment of the present invention, and therefore the specific explanation can be applied to the explanation in the section on the method for preparing cells for disease treatment.

[0040] <Cells obtained by the method of the present invention> The method of the present invention is the above-mentioned method for preparing cells for disease treatment, the method for promoting the migration ability of cells, and the method for promoting the proliferation ability of cells, and the present invention also includes cells obtained by these methods. The cells obtained by the method of the present invention are cells treated with ultrasound by the method of the present invention, and the type of cells and the conditions of ultrasound (frequency, intensity, irradiation time rate, treatment time, etc.) are as described in the section on the method for preparing cells for disease treatment. Specifically, for example, cells treated with ultrasound at a frequency of 40 kilohertz to 1.5 megahertz, mesenchymal stem cells treated with ultrasound at a frequency of 40 kilohertz to 1.5 megahertz, etc. can be mentioned. The cells obtained by the method of the present invention are preferably used as a medicine for disease treatment. The present invention also includes an invention of a pharmaceutical composition for disease treatment containing the cells obtained by the method of the present invention.

[0041] In addition, the cells obtained by the method of the present invention are cells that have been subjected to ultrasonic treatment under specific conditions, and thus have enhanced migration and proliferation capabilities, and enhanced wound repair capabilities as shown in scratch assays, compared to normal cells, but it is not clear what changes have 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 that have been subjected to ultrasonic treatment under the above-mentioned conditions have enhanced migration and proliferation capabilities, and enhanced wound repair capabilities as shown in scratch assays, compared to normal cells, and are suitable for use as cells for treating diseases. It is believed that ultrasonic treatment affects cells in many ways. It is believed that the combination of various characteristic changes, such as promotion of any protein synthesis, inhibition of any protein synthesis, inactivation or activation of existing proteins, effects on cell membranes, and other changes in characteristics, results in the effects of promotion of migration, promotion of proliferation, and enhanced wound repair capabilities as shown in scratch assays. It is believed that it is impossible or impractical to identify all such characteristic changes through further research, measurement, etc. Therefore, the cells of the present invention are specified by the method, as cells obtained by the method of preparing cells for treating a disease, the method of promoting the migration ability of a cell, or the method of promoting the proliferation ability of a cell of the present invention.

[0042] The present invention also includes the culture supernatant of cells obtained by the method of the present invention. Specifically, for example, the culture supernatant of cells treated with ultrasound at a frequency of 40 kilohertz to 1.5 megahertz, the culture supernatant of mesenchymal stem cells treated with ultrasound at a frequency of 40 kilohertz to 1.5 megahertz, etc. The culture supernatant of cells obtained by the method of the present invention is preferably used for the purpose of treating diseases, for cosmetics, and for other purposes. EXAMPLES

[0043] 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.

[0044] Test Example 1: Ultrasonic treatment of adipose-derived mesenchymal stem cells 1 Adipose-derived mesenchymal stem cells (hereinafter referred to as "ADMSCs") were cultured at 6,000 cells / cm using mesenchymal stem cell medium (Growth medium 2, PROMOCELL). 2 After seeding, the culture vessel in which the ADMSCs were cultured was placed in an ultrasonic generator (Opharmic) and ultrasonic treatment was performed from the bottom of the culture vessel, either on the third day only (single irradiation) or on the second and third days (two irradiations). The frequencies were 40 kilohertz (KHz) and 1 megahertz (MHz), and the intensity was 30 mW / cm. 2 The irradiation time rate (duty cycle) was 20%, and the treatment time was 2, 5, and 10 minutes. The control group was untreated with ultrasound (CNTL). The proliferation ability of the cells after ultrasound treatment was evaluated. Four days after the final ultrasound treatment, the cells were subcultured and the cell count was measured. First, the cell culture supernatant was removed, the cells were washed with PBS, and TrpLE was added to detach the cells. After centrifugation, the cells were suspended again in the medium, and about 10 μL was taken, and the cell count was measured using trypan blue staining solution. After the measurement, the cells were seeded into a new culture vessel.

[0045] At 40 kilohertz (KHz), a decrease in proliferation ability was observed under all conditions (Figure 1), whereas at 1 megahertz (MHz), an increase in proliferation ability was observed (Figure 2).

[0046] Test Example 2: Ultrasonic treatment of adipose-derived mesenchymal stem cells 2 ADMSCs were cultured in the same manner as in Test Example 1, and ultrasonic treatment was performed after 1 and 2 days. The frequency was 1.5 megahertz (MHz), and the intensity was 30 mW / cm. 2 The irradiation time rate (duty cycle) was 20%, and the treatment time was 2 minutes, 5 minutes, and 10 minutes. The negative control was an untreated cell. The proliferation ability of the cells after the ultrasonic treatment was evaluated in the same manner as in Test Example 1. The results of counting the number of cells are shown in Figure 3.

[0047] As shown in FIG. 3, no effect on proliferation was observed at 1.5 megahertz (MHz).

[0048] Test Example 3: Ultrasonic treatment of adipose-derived mesenchymal stem cells 3 As in Test Example 1, ADMSCs were cultured and subjected to ultrasonic treatment one day later (single irradiation) or both one and two days later (double irradiation). The frequency was 1 megahertz (MHz) and the intensity was 30 mW / cm. 2 The duty cycle was 50%, and the treatment time for the single irradiation group was 10 minutes, and for the double irradiation group, the treatment time was 2 minutes and 5 minutes. The negative control was not treated with ultrasound. The proliferation ability of the cells after ultrasound treatment was evaluated in the same manner as in Test Example 1. The results of counting the number of cells are shown in Figure 4.

[0049] As shown in FIG. 4, an increase in proliferation ability was observed with either type of irradiation.

[0050] Test Example 4: Ultrasonic treatment of adipose-derived mesenchymal stem cells 4 As in Test Example 1, ADMSCs were cultured and sonicated twice, on days 1 and 2, with the seeding date being day 0. The frequency was 1.5 megahertz (MHz) and the intensity was 30 mW / cm. 2 The irradiation time rate (duty cycle) was 50%, and the treatment time was 10 minutes. The negative control was an untreated cell. The proliferation ability of the cells after the ultrasonic treatment (3 days after seeding) was evaluated in the same manner as in Test Example 1. The results of counting the number of cells are shown in FIG. 5.

[0051] As shown in FIG. 5, no effect on proliferation was observed even at 1.5 megahertz (MHz) and an irradiation time rate of 50%.

[0052] Test Example 5: Scratch assay for sonicated ADMSCs As in Test Example 1, ADMSCs were cultured and sonicated twice, on days 1 and 2, with the seeding date being day 0. The frequency was 1.5 megahertz (MHz) and the intensity was 30 mW / cm. 2 The irradiation time rate (duty cycle) was 50%, and the treatment time was 5 and 10 minutes. The ultrasonically treated ADMSCs were cultured at 15,000 cells / cm. 2The cells were seeded in a φ35 mm culture vessel using mesenchymal stem cell medium (PROMOCELL). After confirming that the cells were confluent, the center of the culture vessel in which the ADMSCs were cultured was scratched in a straight line with a 200 μL pipette tip to create a gap. The cells were then washed twice with PBS, and 2 mL of GM2 medium (PROMOCELL) was added and cultured at 37°C with a carbon dioxide concentration of 5%. The cells were observed under a microscope 0, 4, 9, and 24 hours after the gap was created, and images were taken. The width of the gap in each cell was measured 9 hours later. The results are shown in Figures 6 and 7.

[0053] As shown in Figures 6 and 7, ultrasonic treatment enhanced the degree of wound repair as demonstrated by the scratch assay.

[0054] Test Example 6: Migration test for sonicated ADMSCs As in Test Example 1, ADMSCs were cultured and sonicated twice on days 1 and 2, with the sowing date being designated as day 0. 3,000 cells each were sowed into a 24-well plate Transwell culture vessel (Corning) using mesenchymal stem cell medium (PROMOCELL). After 24 hours, the cells were washed with PBS, and 4% paraformaldehyde was added to fix the cells that had migrated to the underside of the Transwell. These cells were stained with DAPI. The number of cells was counted under a fluorescent microscope, and the average values ​​of the three measurements were compared. Fluorescent micrographs were also taken. Note that the sonication was performed at a frequency of 1.5 megahertz (MHz) and an intensity of 30 mW / cm. 2 The irradiation time rate (duty cycle) was set to 20% or 50%, and the treatment time was set to 2 minutes, 5 minutes, and 10 minutes. The results are shown in Figures 8 and 9.

[0055] As shown in Figures 8 and 9, under all irradiation conditions, the number of cells migrating to the underside of the Transwell increased compared to the non-irradiated control, indicating that ultrasonic treatment enhanced the cell migration ability.

[0056] Although data is not shown, ultrasonic treatment under all conditions was found to increase the expression level of migration-related genes, with a tendency for this to be particularly high at a frequency of 1.5 megahertz (MHz).

[0057] Test Example 8: Subculture of ADMSCs subjected to ultrasonic treatment ADMSCs that had been subjected to ultrasonic treatment in the same manner as in Test Example 1 were detached using trypsin, subcultured for one passage, and cultured in mesenchymal stem cell medium (PROMOCELL) to obtain subcultured cells. The frequency of ultrasonic treatment was 1.5 megahertz (MHz), and the intensity was 30 mW / cm. 2 The irradiation time rate (duty cycle) was set to 50%, and the treatment time was set to 5 minutes.

[0058] Test Example 9: Migration ability test of subcultured cells A migration test was carried out on the subcultured cells obtained in Test Example 8 in the same manner as in Test Example 6. After 24 hours, the number of cells that had migrated to the underside of the Transwell was counted, and the average values ​​of the results of three measurements were compared. Fluorescence microscopic photographs were also taken. The results are shown in Figures 10 and 11. The ultrasound untreated group is indicated as CNTL, and the ultrasound treated group is indicated as US.

[0059] As shown in Figures 10 and 11, ultrasonic treatment increased the number of cells that migrated to the lower surface of the Transwell, even in the subcultured cells.

[0060] Test Example 10: Scratch assay of subcultured cells For the subcultured cells obtained in Test Example 8, the center of the culture vessel in which ADMSCs were cultured was scratched in a straight line to create a gap, and the cells were observed under a microscope 0, 4, 9, and 24 hours after the start of culture, and images were obtained. In addition, the width of the gap in each cell was measured after 9 hours. The results are shown in Figures 12 and 13. The ultrasound untreated group was indicated as CNTL, and the ultrasound treated group was indicated as US.

[0061] Ultrasonic treatment enhanced the degree of wound repair as shown by scratch assay even in subcultured cells (Figures 12 and 13).

[0062] Test Example 11: Freezing and culturing of sonicated ADMSCs ADMSCs that had been subjected to ultrasonic treatment in the same manner as in Example 8 were detached using trypsin and centrifuged to obtain cell precipitates. After removing the supernatant, an appropriate amount of cell freezing preservation solution (Lonza) was added to suspend the cells. The cell suspension was dispensed into cryotubes and stored at -80°C in a freezer, then transferred to the gas phase above liquid nitrogen for continued storage. The ultrasonic-treated frozen cells were put to sleep and cultured in mesenchymal stem cell medium (Promocell) to obtain ultrasonic-treated frozen cultured cells.

[0063] Example 12: Migration ability test of sonicated frozen cultured cells The ultrasonically treated frozen cultured cells obtained in Example 11 were subjected to a migration test in the same manner as in Example 6. That is, 24 hours after seeding in the Transwell culture vessel, the number of cells that had migrated to the underside of the Transwell was counted, and the average values ​​of the results of three measurements were compared. The results are shown in Figures 14 and 15.

[0064] The ultrasound untreated group was designated as CNTL, and the ultrasound treated group was designated as US. Even in the case of frozen-cultured cells, ultrasonic treatment resulted in a greater number of cells migrating to the underside of the Transwell compared to untreated frozen-cultured cells.

[0065] Test Example 13: Scratch assay of sonicated frozen cultured cells For the ultrasonically treated frozen cultured cells obtained in Example 11, the center of the culture vessel in which the cells were cultured was scratched in a straight line to create a gap, and the cells were observed under a microscope 0, 4, 9, and 24 hours after the start of culture, and images were obtained. The width of the gap in each cell after 9 hours was measured. The results are shown in Figures 16 and 17. The ultrasonically untreated group is indicated as CNTL, and the ultrasonically treated group is indicated as US.

[0066] Ultrasonic treatment increased the cell migration rate after scratching and the wound repair rate as shown by scratch assay, even in frozen cultured cells (Figures 16 and 17). [Industrial Applicability]

[0067] According to the present invention, a novel method for promoting the migration ability of cells and cells with promoted migration ability can be provided. Also, according to the present invention, a method for promoting the proliferation ability of cells and cells with promoted proliferation ability can be provided. Therefore, according to the present invention, since cells with excellent migration ability and cells with promoted proliferation ability can be obtained, it is believed that a large number of cells can be accumulated at disease sites such as inflammatory tissues and tumor tissues, and the effect of disease treatment by cells can be improved.

Claims

1. A method for preparing cells for the treatment of a disease, comprising subjecting cells to ultrasonic treatment.

2. 2. The method for preparing cells for the treatment of a disease according to claim 1, wherein the frequency in the ultrasonic treatment is from 30 kilohertz to 2.0 megahertz.

3. The method for preparing cells for disease treatment according to claim 1 or 2, wherein the cells are mesenchymal stem cells.

4. A method for promoting cell migration, comprising subjecting cells to ultrasonic treatment.

5. 5. The method for promoting cell migration according to claim 4, wherein the frequency in the ultrasonic treatment is from 30 kilohertz to 2.0 megahertz.

6. The method for promoting cell migration according to claim 4 or 5, wherein the cells are mesenchymal stem cells.

7. A method for promoting cell proliferation, comprising subjecting cells to ultrasonic treatment.

8. 8. The method for promoting cell proliferation according to claim 7, wherein the frequency in the ultrasonic treatment is from 30 kilohertz to 2.0 megahertz.

9. The method for promoting cell proliferation according to claim 7 or 8, wherein the cells are mesenchymal stem cells.

10. Cells treated with ultrasound at frequencies between 30 kilohertz and 2.0 megahertz.

11. Mesenchymal stem cells treated with ultrasound at frequencies between 30 kilohertz and 2.0 megahertz.

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