Mesenchymal stem cells and medium for mesenchymal stem cells

Abstract

An object of the present invention is to provide a mesenchymal stem cell having excellent cell adhesiveness and capable of obtaining a sufficient cell proliferation rate without requiring a serum medium.SOLUTION: The present invention relates to a mesenchymal stem cell in which a production amount of a granulocyte colony-stimulating factor (G-CSF) is enhanced. The present invention also includes a mesenchymal stem cell in which a production amount of at least one selected from the group consisting of eotaxin, fractalkine, GRO, MCP-3, and VEGF is enhanced.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to mesenchymal stem cells and a medium for mesenchymal stem cells. [Background technology]

[0002] In recent years, cell culture of tissues, cells, fertilized eggs, and the like from various parts of the human body has been put to practical use, and the cultured cells are used in regenerative medicine and other applications. One such example is mesenchymal stem cells. Mesenchymal stem cells are multipotent progenitor cells that were first isolated from bone marrow by Friedenstein (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 Patent Documents 1 and 2). Recently, it has been discovered that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, and the like. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

[0003] Serum media have traditionally been used for culturing mesenchymal stem cells. In cell culture, serum is an important factor as a source of growth factors, adhesion factors, hormones, lipids, and minerals. However, serum media, such as fetal bovine serum, are used, but they pose the risk of contamination with viruses and bacteria due to animal serum. Furthermore, there are problems with variations in serum lot by lot, making it difficult to obtain consistent results. Therefore, efforts have been made to develop serum-free media that replace serum with appropriate nutritional and hormonal components and avoid the problems associated with using animal serum. However, these have presented problems, such as slower cell growth rates than those achieved using serum media.

[0004] Furthermore, when mesenchymal stem cells are cultured in conventional serum-free medium, the cells have poor adhesive properties, so the cell adhesion surface of the culture vessel must be coated with a coating agent such as collagen, laminin, or fibronectin, which is time-consuming and expensive. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-157263 [Patent Document 2] Special Publication No. 2012-508733 [Non-patent literature]

[0006] [Non-Patent Document 1] Pittenger FMet al.Science ,(1999),284,pp.143-147 Summary of the Invention [Problem to be solved by the invention]

[0007] In light of the above-mentioned circumstances, an object of the present invention is to provide mesenchymal stem cells that have excellent cell adhesiveness, do not require a serum medium, and can achieve a sufficient cell proliferation rate. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that mesenchymal stem (stromal) cells (MSCs) with enhanced production of granulocyte colony stimulating factor (G-CSF) require a short time for adhesion and have a high cell proliferation rate, and thus completed the present invention. According to the present invention, mesenchymal stem cells with excellent cell adhesiveness and a high cell proliferation rate can be provided. That is, the gist of the present invention is as follows.

[0009] [1] Mesenchymal stem cells are characterized by increased production of granulocyte colony-stimulating factor (G-CSF). [2] A mesenchymal stem cell characterized by enhanced production of at least one selected from the group consisting of eotaxin, fractalkine, GRO, MCP-3, and VEGF. [3] Mesenchymal stem cells according to [1] or [2], which are derived from adipose tissue, umbilical cord tissue, or bone marrow tissue. [4] A cell culture medium for inducing mesenchymal stem cells according to any one of [1] to [3]. [Effects of the Invention]

[0010] According to the present invention, mesenchymal stem cells having excellent cell adhesiveness and a high cell proliferation rate can be provided. The mesenchymal stem cells of the present invention have excellent adhesiveness and can proliferate at a high rate in any container, even without the use of serum during culture. Furthermore, the mesenchymal stem cells of the present invention reduce the mRNA expression of CCL2, TNF, and PDGFB in macrophages, and also have the effect of suppressing the proliferation of activated T cells, thereby exerting a significant anti-inflammatory effect. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the relationship between the time required for cell adhesion and the amount of G-CSF secreted in mesenchymal stem cells. [Figure 2] FIG. 2 shows the relationship between cell proliferation activity and the amount of G-CSF secreted in mesenchymal stem cells. [Figure 3] FIG. 3 shows the PostPDL of adipose-derived mesenchymal stem cells cultured under each medium condition. [Figure 4] FIG. 4 shows the PostPDL of adipose-derived mesenchymal stem cells cultured under each medium condition. [Figure 5] FIG. 5 shows the PostPDL of umbilical cord-derived mesenchymal stem cells cultured under each medium condition. [Figure 6] FIG. 6 shows the PostPDL of umbilical cord-derived mesenchymal stem cells cultured under each medium condition. [Figure 7]FIG. 7 shows the PostPDL of bone marrow-derived mesenchymal stem cells cultured under each medium condition. [Figure 8] FIG. 8 is a micrograph of human adipose-derived stem cells (ADSCs) cultured in R medium. [Figure 9] FIG. 9 is a micrograph of human adipose-derived stem cells (ADSCs) cultured in MC medium. [Figure 10] FIG. 10 is a micrograph of human adipose-derived stem cells (ADSCs) cultured in SP medium. [Figure 11] FIG. 11 shows the suppression rate of CCL2 mRNA expression level in THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 12] FIG. 12 shows the suppression rate of TNF mRNA expression level in THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 13] FIG. 13 shows the suppression rate of PDGFB mRNA expression level in THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 14] FIG. 14 shows the rate of suppression of T cell proliferation by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 15] FIG. 15 shows the rate of suppression of T cell proliferation by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 16] FIG. 16 shows the rate of suppression of T cell proliferation by co-culture with human umbilical cord-derived mesenchymal stem cells. [Figure 17] FIG. 17 shows the rate of suppression of T cell proliferation by co-culture with human umbilical cord-derived mesenchymal stem cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] The mesenchymal stem cells of the present invention will be described in detail below.

[0013] [Mesenchymal stem cells] The mesenchymal stem cells of the present invention are characterized by enhanced production of granulocyte colony-stimulating factor (G-CSF). The mesenchymal stem cells of the present invention are also characterized by enhanced production of at least one selected from the group consisting of eotaxin, fractalkine, GRO, MCP-3, and VEGF.

[0014] Granulocyte colony-stimulating factor (G-CSF) is a growth factor that specifically induces hematopoietic stem cells to differentiate into neutrophils (granulocytes). Eotaxin is a CC chemokine that selectively confers high chemotaxis to eosinophils. Fractalkine is a membrane-bound chemokine that possesses both chemokine and cell adhesion molecule activities and is expressed on activated vascular endothelial cells. GRO, also known as CXCL1, is a chemokine that is thought to have mitogenic functions and neutrophil chemoattractants. MCP-3 (Monocyte Chemotactic Protein-3), also known as monocyte-specific cytokine-3, is a CC chemokine that acts as a chemotactic factor for monocytes, macrophages, etc. Vascular endothelial growth factor (VEGF) is a protein that promotes angiogenesis, and has the effects of promoting angiogenesis processes including the proliferation of vascular endothelial cells and enhancing vascular permeability.

[0015] In the present invention, "enhanced production of factors such as granulocyte colony-stimulating factor (G-CSF), eotaxin, fractalkine, GRO, MCP-3, and VEGF" includes high mRNA expression levels of the factors, high protein production levels of the factors, high secretion levels of the factors, or any combination thereof. Furthermore, the mesenchymal stem cells of the present invention may be those that have enhanced production levels of the above factors compared to other cells. Specifically, the mesenchymal stem cells of the present invention may be those that have higher gene expression levels, protein production levels, or secretion levels of the above factors compared to mesenchymal stem cells obtained under conventional culture conditions (e.g., culture in 10% FBS-containing DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium).

[0016] Enhanced production of each of the above factors preferably means that the mRNA expression level, protein production level, and secretion level are 120% or more, more preferably 300% or more, even more preferably 1500% or more, particularly preferably 3000% or more, and most preferably 10000% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0017] Specifically, enhanced G-CSF production preferably means that the production amount is 500% or more, more preferably 1000% or more, even more preferably 5000% or more, and particularly preferably 15000% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0018] The term "enhanced eotaxin production" preferably means that the production amount is 200% or more, more preferably 500% or more, even more preferably 1000% or more, and particularly preferably 3000% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0019] The term "enhanced fractalkine production" preferably means that the production amount is 110% or more, more preferably 120% or more, even more preferably 130% or more, and particularly preferably 140% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0020] Enhanced GRO production preferably means that the production amount is 120% or more, more preferably 300% or more, even more preferably 500% or more, and particularly preferably 1500% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0021] Enhanced MCP-3 production preferably means that the production amount is 120% or more, more preferably 150% or more, even more preferably 200% or more, and particularly preferably 300% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0022] Enhanced VEGF production preferably means that the production amount is 110% or more, more preferably 120% or more, even more preferably 130% or more, and particularly preferably 140% or more, compared to mesenchymal stem cells obtained under conventional culture conditions.

[0023] 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, 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, and adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are more preferred.

[0024] In the present invention, mesenchymal stem cells may be derived from human, horse, cow, sheep, pig, dog, cat, rabbit, mouse, or rat.

[0025] In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells, in which at least 20% or more, preferably 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 96% or more, 97% or more, 98% or more, or 99% or more are mesenchymal stem cells.

[0026] In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically removing or aspirating subcutaneous fat from a mammal.

[0027] In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells.

[0028] In the present invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

[0029] (Method for preparing mesenchymal stem cells) The method for preparing the mesenchymal stem cells of the present invention is not particularly limited, but they can be prepared, for example, as follows. Specifically, mesenchymal stem cells can be isolated from tissues such as adipose tissue, umbilical cord, and bone marrow according to methods known to those skilled in the art, and then cultured in a specific medium to induce mesenchymal stem cells with enhanced production of various factors, thereby obtaining the mesenchymal stem cells of the present invention. In the cell population obtained by this induction, preferably 50% or more of the cell population are the cells of the present invention, more preferably 70% or more are the cells of the present invention, even more preferably 80% or more are the cells of the present invention, and particularly preferably 90% or more are the cells of the present invention, and most preferably a substantially homogeneous cell population of the cells of the present invention.

[0030] From the viewpoint of using the mesenchymal stem cells of the present invention as a cell medicine, it is preferable to use a xeno-free medium that does not contain xenogeneic components such as serum. Among such media, from the viewpoint of being highly effective in converting mesenchymal stem cells into mesenchymal stem cells of the present invention in which the production of specific factors such as G-CSF is enhanced, the viscosity is in the range of 1.00 to 1.20, preferably 1.05 to 1.10. Furthermore, the osmotic pressure of the medium is in the range of 1.00 to 1.10, preferably 1.04 to 1.05. Media that are effective in converting mesenchymal stem cells into mesenchymal stem cells of the present invention in which the production of specific factors such as G-CSF is enhanced are within the scope of the present invention.

[0031] To confirm that the selected cells are adipose tissue-derived mesenchymal stem cells of the present invention, surface antigens may be analyzed by conventional methods such as flow cytometry. Furthermore, the ability to differentiate into each cell lineage may be examined, and such differentiation can be carried out by conventional methods.

[0032] The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells that have the following properties in addition to enhanced production of specific factors such as G-CSF: (1) Adhesion to plastic under standard culture conditions. (2) Surface antigens CD44, CD73, and CD90 are positive, and CD31 and CD45 are negative. (3) Under certain culture conditions, they can be differentiated into osteocytes, adipocytes, and chondrocytes.

[0033] The mesenchymal stem cells of the present invention may be in any state, for example, cells recovered by detaching cells during culture, or cells frozen in a cryopreservation solution. [Example]

[0034] 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.

[0035] [Example 1: Cell culture 1] Human umbilical cord-derived mesenchymal stem cells (Promo Cell, hereafter referred to as "hUCMSC") were cultured in a T75 flask for adherent cells (Corning) at a density of 5000 cells / cm. 2The cells were seeded in 10% FBS DMEM / F12 medium (Sigma, hereafter referred to as "DMEM / F12 medium"), R:Stem serum-free medium for mesenchymal stem cells (Rohto, hereafter referred to as "R medium"), StemFit For Mesenchymal Stem Cell (Ajinomoto, hereafter referred to as "SF medium"), and STEMPRO® MSC SFM (Thermo Fisher Scientific, hereafter referred to as "SP medium") for 3 days (37°C, 5% CO2). Then, the cells were passaged using cell detachment solution, and the supernatant was collected after an additional 4 days of culture (37°C, 5% CO2). Granulocyte colony-stimulating factor (G-CSF) in cell supernatants was measured using a MAGPIX® system (Merck Millipore) with a Human Cytokine / Chemokine Magnetic Bead Panel (EMD Millipore, Cat.: HCYTOMAG-60K) in a multiplex assay. The amount of G-CSF in the supernatants of cells cultured in each medium was calculated relative to the G-CSF content in the supernatant of cells cultured in DMEM / F12 medium, which was defined as 100%. The results are shown in Table 1.

[0036] Furthermore, hUCMSCs were seeded in DMEM / F12 medium, R medium, SF medium, and SP medium, and the time until the cells became stationary (adhesion) was measured using a live cell imaging system (SI8000, Sony Corporation). The results are shown in Table 1.

[0037] Furthermore, hUCMSCs were seeded in DMEM / F12 medium, R medium, SF medium, and SP medium, respectively, and cultured for 2 days (37°C, 5% CO2), and cell proliferation activity was evaluated using WST-8 (Cell Counting Kit-8) (Table 1).

[0038] [Table 1]

[0039] As shown in Table 1, the supernatant of mesenchymal stem cells cultured in R medium was found to contain a higher amount of G-CSF than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, the supernatant of mesenchymal stem cells cultured in other serum-free media was found to contain a lower amount of G-CSF than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium.

[0040] Furthermore, mesenchymal stem cells cultured in R medium took less time to adhere to the culture vessel than mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, mesenchymal stem cells cultured in other serum-free media took longer to adhere to the culture vessel than mesenchymal stem cells cultured in DMEM / F12 medium.

[0041] Furthermore, mesenchymal stem cells cultured in R medium had higher cell proliferation activity than mesenchymal stem cells cultured in DMEM / F12 medium, whereas mesenchymal stem cells cultured in other serum-free media had lower cell proliferation activity than mesenchymal stem cells cultured in DMEM / F12 medium.

[0042] These results demonstrate that mesenchymal stem cells cultured in R medium secrete large amounts of G-CSF, require less time for cell adhesion, and have high cell proliferation activity.

[0043] Furthermore, for the cells cultured in different media, the relationship between the time required for cell adhesion and the amount of G-CSF secreted, and the relationship between cell proliferation activity and the amount of G-CSF secreted, are shown in Figures 1 and 2, respectively. The more G-CSF secreted by the cells, the shorter the time required for cell adhesion (Figure 1). The more G-CSF secreted by the cells, the higher the cell proliferation activity (Figure 2).

[0044] [Example 2: Cell culture 2] As in Example 1, hUCMSCs were cultured in a T75 flask for adherent cells (Corning) at 5000 cells / cm. 2Cells were seeded at 100°C and cultured in DMEM / F12, R, or SP media for 3 days at 37°C and 5% CO2. They were then passaged using cell detachment solution and cultured for an additional 4 days at 37°C and 5% CO2, after which the supernatants were collected. Eotaxin and fractalkine in the cell supernatants were measured using a MAGPIX® system (Merck Millipore) with a Human Cytokine / Chemokine Magnetic Bead Panel (EMD Millipore, Cat.: HCYTOMAG-60K) in a multiplex assay. The amounts of eotaxin and fractalkine in the supernatants of cells cultured in each medium were calculated relative to the eotaxin and fractalkine content in the supernatants of cells cultured in DMEM / F12 medium (Table 2).

[0045] Furthermore, hUCMSCs were seeded in DMEM / F2 medium, R medium, and SP medium, respectively, and the migration distance was measured using a live cell imaging system (SI8000, Sony) (Table 2).

[0046] These results demonstrate that mesenchymal stem cells cultured in R medium secrete large amounts of eotaxin and fractalkine and migrate long distances.

[0047] [Table 2]

[0048] As shown in Table 2, the supernatant of mesenchymal stem cells cultured in R medium was found to contain higher amounts of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, the supernatant of mesenchymal stem cells cultured in SP medium contained similar amounts of eotaxin and fractalkine to the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. Furthermore, mesenchymal stem cells cultured in R medium had a significantly longer migration distance than mesenchymal stem cells cultured in DMEM / F12 medium.

[0049] [Example 3: Cell culture 3] As in Example 1, hUCMSCs were cultured in a T75 flask for adherent cells (Corning) at 5000 cells / cm. 2 Cells were seeded at 100°C and cultured in DMEM / F12 medium, R medium, or MesenPRO RS™ Medium (Thermo Fisher Scientific, hereafter referred to as "MP medium") for 3 days at 37°C and 5% CO2. They were then passaged using cell detachment solution, and after an additional 4 days of culture at 37°C and 5% CO2, the supernatant was collected. Eotaxin and fractalkine in the cell supernatant were measured using a MAGPIX® system (Merck Millipore) with a Human Cytokine / Chemokine Magnetic Bead Panel (EMD Millipore, Cat.: HCYTOMAG-60K) in a multiplex assay. The amounts of eotaxin and fractalkine in the supernatant of cells cultured in each medium were calculated relative to the eotaxin and fractalkine content in the supernatant of cells cultured in DMEM / F12 medium (Table 3).

[0050] In addition, hUCMSCs were seeded in DMEM / F2 medium, R medium, and MP medium, respectively, and the time until the cells became stationary (adhesion) and the migration distance were measured using a live cell imaging system (SI8000, Sony) (Table 3).

[0051] Furthermore, hUCMSCs were seeded in DMEM / F12 medium, R medium, and MP medium, respectively, and then cultured for 2 days (37°C, 5% CO2), and cell proliferation activity was evaluated using WST-8 (Cell Counting Kit-8) (Table 3).

[0052] [Table 3]

[0053] As shown in Table 3, the supernatant of mesenchymal stem cells cultured in R medium was found to contain higher amounts of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, the supernatant of mesenchymal stem cells cultured in MS medium contained similar amounts of eotaxin and fractalkine to the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium.

[0054] Furthermore, mesenchymal stem cells cultured in R medium took significantly less time to adhere to the container and had higher cell proliferation activity than mesenchymal stem cells cultured in DMEM / F12 medium, whereas mesenchymal stem cells cultured in MS medium were comparable to those cultured in DMEM / F12 medium.

[0055] From the above results, it was revealed that mesenchymal stem cells cultured in R medium secreted large amounts of eotaxin and fractalkine, migrated long distances, and had high cell proliferation activity.

[0056] [Example 4: Cell culture 4] As in Example 1, hUCMSCs were cultured in a T75 flask for adherent cells (Corning) at 5000 cells / cm. 2 Cells were seeded at 100°C and cultured in DMEM / F12, R, SF, SP, or MS media for 3 days at 37°C and 5% CO2. They were then passaged using cell detachment solution and cultured for an additional 4 days at 37°C and 5% CO2. The supernatants were then collected. A multiplex assay was performed using a MAGPIX® system (Merck Millipore) with a Human Cytokine / Chemokine Magnetic Bead Panel (EMD Millipore, Cat.: HCYTOMAG-60K) to measure GRO, Monocyte Chemotactic Protein-3 (MCP-3), and Vascular Endothelial Growth Factor (VEGF). The content of each component in the supernatant of cells cultured in each medium was calculated relative to the content of each component in the supernatant of cells cultured in DMEM / F12 medium (Table 4).

[0057] [Table 4]

[0058] As shown in Table 4, the supernatant of mesenchymal stem cells cultured in R medium contained higher amounts of GRO, MCP-3, and VEGF eotaxin than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, the mesenchymal stem cells cultured in other serum-free media had lower amounts of each factor than the mesenchymal stem cells cultured in DMEM / F12 medium.

[0059] Example 5: Proliferation of adipose-derived mesenchymal stem cells 1 After obtaining consent from human donors, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To disrupt the extracellular matrix and isolate cells, collagenase (Roche Diagnostics) was added (solvent: physiological saline) and the cells were dispersed by shaking at 37°C for 90 minutes. The suspension was then centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the cell precipitate, and the cell suspension was centrifuged at 400g for 5 minutes. After removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto) and seeded into flasks. The cells were cultured at 37°C for several days in 5% CO2. After several days, the cultures were washed with PBS to remove blood cells and residual adipose tissue, and mesenchymal stem cells adhered to the plastic container were obtained. The obtained adipose tissue-derived mesenchymal stem cells were cultured in a T25 flask (Corning) for adherent cells at 5000 cells / cm. 2 The cells were seeded at 1000 x g for 4 days and cultured in DMEM / F12 medium, R medium, SP medium, and MS medium (37°C, 5% CO2). The number of cells was measured after 4, 7, and 11 days of culture to determine PostPDL (Figure 3).

[0060] As shown in FIG. 3, the adipose-derived mesenchymal stem cells cultured in R medium had higher cell proliferation activity than the adipose-derived mesenchymal stem cells cultured in DMEM / F12 medium and SP medium.

[0061] [Example 6: Proliferation of adipose-derived mesenchymal stem cells 2] Human adipose-derived mesenchymal stem cells (Promo Cell) were cultured in a T25 flask (Corning) for adherent cells at 5000 cells / cm. 2 The cells were seeded at 1000 x g / ml and cultured in DMEM / F12 medium and R medium, respectively. The number of cells was measured after 4, 7, and 11 days of culture to determine PostPDL (Figure 4).

[0062] As in Example 5, the adipose-derived mesenchymal stem cells cultured in R medium had higher cell proliferation activity than the adipose-derived mesenchymal stem cells cultured in DMEM / F12.

[0063] Example 7: Proliferation of umbilical cord-derived mesenchymal stem cells 1 Umbilical cords collected with the donor's consent were washed with physiological saline. To disrupt the extracellular matrix and isolate cells, collagenase (Roche Diagnostics) (solvent: physiological saline) was added and the cells were dispersed by shaking at 37°C for 90 minutes. The suspension was then centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the cell precipitate, and the cell suspension was centrifuged at 400g for 5 minutes. After removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto) and seeded into flasks. The cells were cultured at 37°C for several days in 5% CO2. After several days, the culture was washed with PBS to remove blood cells and residual umbilical cord tissue from the culture medium, and mesenchymal stem cells adhered to a plastic container were obtained. The resulting umbilical cord-derived mesenchymal stem cells were placed in T25 flasks for adherent cells (Corning) at 5000 cells / cm. 2 The cells were seeded at 100°C and cultured in DMEM / F12 medium, R medium, SP medium, MS medium, and MesenCult™ MSC Basal Medium (Human) (STEMCELL Technologies, hereafter referred to as "MC medium") at 37°C and 5% CO2. The number of cells was measured after 4, 7, and 11 days of culture to determine PostPDL (Figure 5).

[0064] Umbilical cord-derived mesenchymal stem cells cultured in R medium had higher cell proliferation activity than umbilical cord-derived mesenchymal stem cells cultured in DMEM / F12 medium and other serum-free media.

[0065] Example 8: Proliferation of umbilical cord-derived mesenchymal stem cells 2 Human umbilical cord-derived mesenchymal stem cells (Promo Cell) were cultured in a T25 flask (Corning) for adherent cells at 5000 cells / cm. 2 The cells were seeded at 1000 x g / ml and cultured in DMEM / F12 medium and R medium (37°C, 5% CO2), respectively. The number of cells was measured after 4, 7, 10, and 13 days of culture to determine PostPDL (Figure 6).

[0066] Umbilical cord-derived mesenchymal stem cells cultured in R medium had higher cell proliferation activity than umbilical cord-derived mesenchymal stem cells cultured in DMEM / F12 medium.

[0067] Example 9: Expansion of bone marrow-derived mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (Promo Cell) were cultured in a T25 flask for adherent cells (Thermo Fisher Scientific) at 5000 cells / cm. 2 The cells were seeded at 1000 x g / ml and cultured in DMEM / F12 medium and R medium (37°C, 5% CO2), respectively. The number of cells was measured after 2, 7, 12, and 15 days of culture to determine PostPDL (Figure 7).

[0068] Umbilical cord-derived mesenchymal stem cells cultured in R medium had higher cell proliferation activity than umbilical cord-derived mesenchymal stem cells cultured in DMEM / F12 medium.

[0069] Example 10: Adhesion of adipose-derived mesenchymal stem cells Human adipose-derived stem cells (ADSCs) (LONZA, hereafter referred to as "ADSCs") were cultured in a culture flask for adherent cells (Nunc EasyFlask Cell Culture Flasks, T25, cat: 156367, no coating agent used) at a density of 2000 cells / cm. 2The cells were seeded in R medium, MC medium, and SP medium, respectively, and cultured for 48 hours (37°C, 5% CO2), and the state of cell adhesion was observed under a microscope. Micrographs are shown in Figure 8 (R medium), Figure 9 (MC medium), and Figure 10 (SP medium).

[0070] In R medium, adipocyte-derived stem cells were observed adipocyte-derived stem cells adhering to the flask and elongating, but in other serum-free media, no elongation was observed. From the above, it was confirmed that R medium improves cell adhesion compared to other serum-free media.

[0071] Example 11: Effect on THP-1 macrophages The human acute monocytic leukemia-derived cell line THP-1 (RIKEN BioResource Center, hereafter referred to as "THP-1") was expanded and frozen according to the recommended protocol. On day 9 after THP-1 seeding, the THP-1 suspension was harvested and Phorbol 12-Myristate 13-Acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the THP-1 suspension at a final concentration of 100 nM. 1 mL of the THP-1 suspension was seeded into each well of a 12-well plate (Corning Inc.) and cultured at 37°C and 5% CO2 to induce THP-1 differentiation. hUCMSCs were cultured in DMEM / F12 medium, R medium, SF medium, MP medium, and SP medium, respectively. The cryopreserved cells were then thawed to a viable cell concentration of 8.96 x 10 410% FBS-RPMI medium was added to the wells to achieve a cell density of 100 cells / mL, and LPS-EB (InvivoGen) was added to a final concentration of 1 μg / mL. The 10% FBS-RPMI medium was prepared by adding 50 mL of FBS and 5 mL of Penicillin-Streptomycin Liquid (Thermo Fisher Scientific Inc.) to 445 mL of RPMI-1640 Medium (Thermo Fisher Scientific Inc.). On day 3 after THP-1 differentiation induction, the prepared cells were added to the Transwell inserts and co-cultured for 2 days. Two days after the initiation of co-culture, THP-1 total RNA was collected, and the mRNA expression levels of CCL2, TNF, and PDGFB were measured by quantitative PCR. The percent inhibition was calculated from the mRNA expression levels obtained for each sample using Equation (1). The primers listed in Table 5 were used. The results are shown in Figure 11 (CCL2), Figure 12 (TNF), and Figure 13 (PDGFB). 100 x (1 - (mRNA amount of each sample) / (mRNA amount of LPS(+))) ...Equation (1)

[0072] [Table 5]

[0073] Co-culture of THP-1 macrophages with umbilical cord-derived mesenchymal stem cells suppressed the mRNA expression of CCL2, TNF, and PDGFB. This effect was greatest when umbilical cord-derived mesenchymal stem cells were cultured in R medium.

[0074] [Culture characteristics] The viscosity of DMEM / F12, R, SF, SP, and MS media was measured at 20°C, 100 rpm, and 180 seconds using a low-pressure viscometer (Rotor No. 01, TVE-20L, Toki Sangyo Co., Ltd.). Osmolality and pH were also measured (Table 6). The osmolality ratio was defined as the ratio of the osmolality of the sample to the osmolality of 286 mOsm (0.9 w / v% sodium chloride solution) according to the 17th Revised Japanese Pharmacopoeia. Osmolality was measured using the freezing-point depression method described in the Japanese Pharmacopoeia. The standard solution for measuring the osmolality ratio (0.9 w / v% sodium chloride solution) was Otsuka Saline Injection (Otsuka Pharmaceutical Factory), as specified in the Japanese Pharmacopoeia.

[0075] [Table 6]

[0076] All serum-free media had lower viscosities than DMEM / F12 medium, with R medium having the lowest. SP medium had the same osmotic pressure as DMEM / F12 medium, but the other serum-free media had lower osmotic pressures than DMEM / F12 medium, with R medium having the lowest. These results suggest that R medium may provide strong cell adhesion despite its low viscosity and low osmotic pressure.

[0077] Example 12: Effect on T cells 1 Cryopreserved hUCMSCs were cultured in R medium or SF medium and then thawed and placed in a 24-well plate at 1.33 x 10 5 The cells were seeded at 4.00x10 cells / well and cultured for 15 hours or more. Afterwards, they were stained with carboxyfluorescein succinimidyl ester (5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, hereafter referred to as "CFSE") and stimulated with anti-CD3 / CD28 antibodies. 5The cells were co-cultured with peripheral blood mononuclear cells (PBMCs) at 1000 cells / well for 4 days. After co-culture, PBMCs were collected and the percentage of proliferated T cells was measured using a flow cytometer based on the fluorescence intensity of CFSE staining. CD4+ and CD8+ T cells were analyzed, and these T cells were identified by antibody staining for CD3, CD4, and CD8. The percentage of proliferated T cells was measured by setting a gate at a proliferation rate of approximately 0.5% in the non-anti-CD3 / CD28 antibody-stimulated group, and applying the same gate to the anti-CD3 / CD28 antibody-stimulated test group. Human adipose-derived mesenchymal cells (hADMSCs) were used as a positive control (not shown). Compared to the group stimulated with anti-CD3 / CD28 antibodies but not co-cultured with MSCs (CNTL), the percentage of proliferated CD4+ T cells and CD8+ T cells was reduced in the group co-cultured with hUCMSCs (Figures 14 and 15). Furthermore, the proliferation inhibitory effect of hUCMSCs cultured in SF medium was weaker than that of hUCMSCs cultured in R medium.

[0078] [Example 13: Effect on T cells 2] Cryopreserved hUCMSCs cultured in DMEM / F12 medium were thawed, and the percentage of proliferated T cells was measured in the same manner as in Example 12. Compared to the group stimulated with anti-CD3 / CD28 antibodies and not co-cultured with hUCMSCs (CNTL), the percentage of proliferated CD4+ T cells and CD8+ T cells was reduced in the group co-cultured with hUCMSCs (Figures 16 and 17). Comparison with the positive controls in Examples 12 and 13 revealed that the T cell proliferation inhibitory effect of hUCMSCs cultured in R medium was greater than that of hUCMSCs cultured in DMEM / F12 medium. [Industrial Applicability]

[0079] The present invention provides mesenchymal stem cells that have high cell adhesiveness and rapid cell proliferation. Furthermore, the mesenchymal stem cells of the present invention reduce the mRNA expression of CCL2, TNF, and PDGFB in macrophages and also suppress the proliferation of activated T cells, thereby exerting a significant anti-inflammatory effect.

Claims

1. A mesenchymal stem cell characterized by having enhanced production of at least one selected from the group consisting of eotaxin, fractalkine, GRO, MCP-3 and VEGF.

2. The mesenchymal stem cells according to claim 1, which are derived from adipose tissue, umbilical cord tissue, or bone marrow tissue.

3. A cell culture medium for inducing mesenchymal stem cells according to claim 1 or 2.

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