WO2021192670A1 - Mesenchymal stem cells and medium for mesenchymal stem cells - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
Definitions
- the present invention relates to a mesenchymal stem cell and a medium for mesenchymal stem cells.
- mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells are present in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new therapeutic method for various intractable diseases ( See Patent Documents 1 and 2).
- mesenchymal stem cells may be referred to as stromal cells (Mesenchymal Cell).
- serum medium Conventionally, a serum medium has been used for culturing mesenchymal stem cells.
- serum is an important source of growth factors, adhesion factors, hormones, lipids and minerals.
- serum such as fetal bovine serum
- serum-free media have been developed that can replace the serum with appropriate nutritional and hormonal components to avoid the problem of animal serum use, but the cell growth rate is slower than when the serum medium is used. There was a problem.
- the cell adhesion is weak, so it is necessary to coat the cell adhesion surface of the culture vessel with a coating agent such as collagen, laminin, or fibronectin. It took time and money.
- An object of the present invention is to provide mesenchymal stem cells which are excellent in cell adhesion and can obtain a sufficient cell proliferation rate without the need for a serum medium under the above-mentioned circumstances.
- G-CSF Granulocyte Colony Stimulating Factor
- MSC mesenchymal stem cells
- [1] Mesenchymal stem cells, characterized in that the amount of production (Production) of a granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased.
- G-CSF Granulocyte Colony Stimulating Factor
- a mesenchymal stem cell characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced.
- a cell medium for inducing mesenchymal stem cells according to any one of [1] to [3].
- mesenchymal stem cells having excellent cell adhesion and a high cell proliferation rate.
- the mesenchymal stem cells of the present invention have excellent adhesiveness and can proliferate at a high rate in any container without using serum in culture.
- the mesenchymal stem cells of the present invention have an effect of reducing the mRNA expression of macrophages CCL2, TNF and PDGFB and suppressing the proliferation of activated T cells, and exert a remarkable anti-inflammatory effect.
- FIG. 1 is a diagram showing the relationship between the time required for cell adhesion and the amount of G-CSF secreted in mesenchymal stem cells.
- FIG. 2 is a diagram showing the relationship between cell proliferation activity and the amount of G-CSF secreted in mesenchymal stem cells.
- FIG. 3 is a diagram showing PostPDL of adipose-derived mesenchymal stem cells cultured under each medium condition.
- FIG. 4 is a diagram showing PostPDL of adipose-derived mesenchymal stem cells cultured under each medium condition.
- FIG. 5 is a diagram showing PostPDL of umbilical cord-derived mesenchymal stem cells cultured under each medium condition.
- FIG. 6 is a diagram showing PostPDL of umbilical cord-derived mesenchymal stem cells cultured under each medium condition.
- FIG. 7 is a diagram showing PostPDL of bone marrow-derived mesenchymal stem cells cultured under each medium condition.
- FIG. 8 is a photomicrograph of human adipocyte-derived stem cells (ADSC) cultured in R medium.
- FIG. 9 is a photomicrograph of human adipocyte-derived stem cells (ADSC) cultured in MC medium.
- FIG. 10 is a photomicrograph of human adipocyte-derived stem cells (ADSC) cultured in SP medium.
- FIG. 11 is a diagram showing the suppression rate of CCL2 mRNA expression level of THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 12 is a diagram showing the suppression rate of TNF mRNA expression level of THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 13 is a diagram showing the suppression rate of PDGFB mRNA expression level of THP-1 macrophages by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 14 is a diagram showing the growth inhibition rate of T cells by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 15 is a diagram showing the growth inhibition rate of T cells by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 16 is a diagram showing the growth inhibition rate of T cells by co-culture with human umbilical cord-derived mesenchymal stem cells.
- FIG. 17 is a diagram showing the growth inhibition rate of T cells by co-culture with human umbilical cord-derived mesenchymal stem cells.
- mesenchymal stem cells of the present invention will be described in detail.
- the mesenchymal stem cells of the present invention are characterized in that the production amount of granulocyte colony stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased.
- G-CSF Granulocyte Colony Stimulating Factor
- the mesenchymal stem cells of the present invention are characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced. do.
- G-CSF Granulocyte Colony Stimulating Factor
- EOTAXIN is one of CC chemokines that selectively imparts high chemotaxis to eosinophils.
- Fractalkine is a cell membrane-bound chemokine that has two activities, a chemokine and a cell adhesion molecule, and is expressed on activated vascular endothelial cells.
- GRO is a chemokine, also known as CXCL1, which is considered to be a mitotic function and neutrophil chemoattractant.
- MCP-3 Monocyte Chemotactic Protein-3
- cytokine-3 a CC chemokine also called cytokine-3, which is a chemotaxis factor for monocytes, macrophages and the like.
- Vascular Endothelial Growth Factor (VEGF) is a protein that promotes angiogenesis, and has an effect of promoting angiogenesis including proliferation of vascular endothelial cells and increasing vascular permeability.
- the production amount of each factor such as granulocyte colony stimulating factor (G-CSF), eotaxin (EOTAXIN), fractalkine (FRACTALKINE), GRO, MCP-3, VEGF is increased in cells.
- G-CSF granulocyte colony stimulating factor
- EOTAXIN eotaxin
- FRACTALKINE fractalkine
- GRO GRO
- MCP-3 MCP-3
- VEGF vascular endothelial growth factor
- the mesenchymal stem cell of the present invention may have an increased production amount of each of the above factors as compared with other cells.
- the mesenchymal stem cell of the present invention has conventional culture conditions.
- the gene expression level of each of the above factors is higher. It suffices if the amount of protein produced is large or the amount of each factor secreted is large.
- the increase in the production amount of each of the above factors means that the mRNA expression level, protein production amount, and secretion amount are preferably 120% or more, more preferably 120% or more, as compared with the mesenchymal stem cells obtained under the conventional culture conditions. It means that it is 300% or more, more preferably 1500% or more, particularly preferably 3000% or more, and most preferably 10000% or more.
- the production amount of G-CSF is increased, preferably the production amount is 500% or more, more preferably 1000%, with respect to the mesenchymal stem cells obtained under the conventional culture conditions. Above, it means that it is more preferably 5000% or more, and particularly preferably 15000% or more.
- the amount of eotaxin produced is increased means that the amount of mesenchymal stem cells produced is preferably 200% or more, more preferably 500% or more, still more preferably 1000, with respect to the mesenchymal stem cells obtained under conventional culture conditions. % Or more, particularly preferably 3000% or more.
- the amount of fractalkine produced is increased means that the amount of fractalkine produced is preferably 110% or more, more preferably 120% or more, still more preferably 120% or more, based on the mesenchymal stem cells obtained under conventional culture conditions. It means that it is 130% or more, particularly preferably 140% or more.
- the fact that the amount of GRO produced is increased means that the amount of GRO produced is preferably 120% or more, more preferably 300% or more, still more preferably 500%, based on the mesenchymal stem cells obtained under conventional culture conditions.
- the above is particularly preferably 1500% or more.
- the increase in the production amount of MCP-3 means that the production amount is preferably 120% or more, more preferably 150% or more, still more preferably 150% or more, based on the mesenchymal stem cells obtained under the conventional culture conditions. It means that it is 200% or more, particularly preferably 300% or more.
- the fact that the amount of VEGF produced is increased means that the amount of VEGF produced is preferably 110% or more, more preferably 120% or more, still more preferably 130%, based on the mesenchymal stem cells obtained under conventional culture conditions.
- the above is particularly preferably 140% or more.
- the mesenchymal stem cell has an ability to differentiate into one or more cells belonging to the mesenchymal system (osteocytes, myocardial cells, chondrocytes, tendon cells, adipocytes, etc.), and maintains the ability. It means a cell that can proliferate.
- mesenchymal stem cell used in the present invention means the same cell as a stromal cell, and does not particularly distinguish between the two. It may also be simply referred to as mesenchymal cells.
- tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, cord blood, endometrial membrane, placenta, amniotic membrane, chorion, decidua, dermatitis, skeletal muscle, bone membrane, tooth follicles, and periodontal ligament.
- tissues containing mesenchymal stem cells include dental pulp and tooth germ.
- adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue, and may be referred to as adipose tissue-derived stromal cells.
- adipose tissue-derived mesenchymal stem cells 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 preferable, and adipose tissue-derived mesenchymal stem cells, Umbilical band-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells are more preferable.
- Examples of the species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats.
- the mesenchymal stem cell means an arbitrary cell population including the mesenchymal stem cell.
- the cell population is 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.
- adipose tissue means a tissue containing stromal cells including adipocytes and microvascular cells, and is, for example, a tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal.
- the umbilical cord is a white tubular tissue that connects the fetal body and the placenta, and is composed of umbilical veins, umbilical arteries, glue-like tissues (Wharton's Jelly), umbilical cord matrix itself, and the like, and is composed of mesenchymal stem cells. Including a lot.
- the bone marrow is a parenchyma that fills the lumen of bone and is a hematopoietic organ.
- Bone marrow fluid exists in the bone marrow, and the cells existing in it are called bone marrow cells.
- Bone marrow cells include mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells and the like, as well as red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like.
- Bone marrow cells can be harvested from, for example, human ilium, long bone, or other bone.
- the method for preparing mesenchymal stem cells of the present invention is not particularly limited, but can be prepared as follows, for example. That is, a mesenchymal system in which the production amount of each factor was enhanced by separating mesenchymal stem cells from tissues such as adipose tissue, umbilical cord, and bone marrow according to a method known to those skilled in the art and culturing them in a specific medium. By inducing stem cells, the mesenchymal stem cells of the present invention can be obtained.
- 50% or more of the cell population is preferably the cells of the present invention, 70% or more is more preferably the cells of the present invention, and 80% or more is the cells of the present invention. More preferably, 90% or more of the cells of the present invention are particularly preferable, and substantially a uniform cell population of the cells of the present invention is most preferable.
- the mesenchymal stem cells of the present invention As a cell medicine, it is preferable to use a medium containing no heterologous components such as serum (Xenofree). Even in such a medium, the mesenchymal stem cells have a viscosity of 1.00 to 1.00 to higher, from the viewpoint of excellent effect of converting the mesenchymal stem cells into the mesenchymal stem cells of the present invention in which the production amount of a specific factor such as G-CSF is enhanced. It is in the range of 1.20, preferably 1.05 to 1.10. The osmotic pressure of the medium is in the range of 1.00 to 1.10, preferably 1.04 to 1.05. The medium effective for converting the mesenchymal stem cells into the mesenchymal stem cells of the present invention in which the production of specific factors such as G-CSF is enhanced is within the scope of the present invention.
- a medium containing no heterologous components such as serum (Xenofree).
- the mesenchymal stem cells have a visco
- the surface antigen may be analyzed by a conventional method using flow cytometry or the like.
- the ability to differentiate into each cell lineage may be tested, and such differentiation can be performed by conventional methods.
- the mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics in addition to increasing the production of specific factors such as G-CSF. ; (1) Shows adhesiveness to plastic under culture conditions in standard medium. (2) The surface antigens CD44, CD73 and CD90 are positive, and CD31 and CD45 are negative. (3) It can be differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.
- the mesenchymal stem cell of the present invention may be a cell in any state, for example, a cell recovered by exfoliating a cell in culture, or a cell frozen in a cryopreservation solution. good.
- Example 1 Cell culture 1
- Human umbilical mesenchymal stem cells (Promo Cell, hereinafter referred to as "hUCMSC") were seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and each was seeded with 10% FBS DMEM / F12 medium (Sigma).
- R Stem mesenchymal stem cell serum-free medium (Rohto, hereinafter referred to as “R medium”), StemFit For Mesenchymal Stem Cell (Ajinomoto, hereinafter referred to as “SF medium”) ) And STEMPRO® MSC SFM (Thermo Fisher Scientific, hereinafter referred to as "SP medium”) were cultured for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected.
- R medium Stem mesenchymal stem cell serum-free medium
- SF medium StemFit For Mesenchymal Stem Cell
- SP medium STEMPRO® MSC SFM
- G-CSF Granulocyte colony stimulating factor
- hUCMSC was inoculated into DMEM / F12 medium, R medium, SF medium and SP medium, respectively, and the time until the cells became stationary (adhered) was measured using a live cell imaging system (SI8000, SONY). The results are shown in Table 1.
- hUCMSC is inoculated into DMEM / F12 medium, R medium, SF medium and SP medium, respectively, and then cultured for 2 days (37 ° C., 5% CO 2 ) to increase the cell proliferation activity of WST-8 (Cell Counting Kit-8). ) (Table 1).
- the mesenchymal stem cells cultured in R medium had a shorter time to adhere to the culture vessel than the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in other serum-free medium took a longer time to adhere to the culture vessel than the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in R medium had higher cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in other serum-free mediums had lower cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium.
- FIG. 1 For cells using different media used for the above culture, the relationship between the time required for cell adhesion and the amount of G-CSF secreted, and the relationship between the cell proliferation activity and the amount of G-CSF secreted are shown in FIG. And shown in FIG. The larger the amount of G-CSF secreted, the shorter the time required for cell adhesion (Fig. 1). The cells with higher G-CSF secretion had higher cell proliferation activity (Fig. 2).
- Example 2 Cell culture 2
- hUCMSC was seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium and SP medium for 3 days (37 ° C., 5% CO 2), respectively. ) was performed. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected.
- hUCMSC was inoculated into DMEM / F2 medium, R medium and SP medium, respectively, and then the migration distance was measured using a live cell imaging system (SI8000, SONY) (Table 2).
- the supernatant of mesenchymal stem cells cultured in R medium contained a higher content of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field.
- the supernatant of the mesenchymal stem cells cultured in SP medium was similar to the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in R medium had a significantly longer migration distance than the mesenchymal stem cells cultured in DMEM / F12 medium.
- Example 3 Cell culture 3
- hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and MesenPRO RS TM Medium (Thermo Fisher Scientific), hereinafter ". It was cultured in MP medium) for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected.
- hUCMSC was seeded in DMEM / F12 medium, R medium and MP medium, respectively, and then cultured for 2 days (37 ° C., 5% CO 2 ) to increase cell proliferation activity in WST-8 (Cell Counting Kit-8). Evaluated (Table 3).
- the supernatant of mesenchymal stem cells cultured in R medium contained a higher content of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field.
- the supernatant of the mesenchymal stem cells cultured in MS medium was similar to the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in R medium had a significantly shorter time to adhere to the container and higher cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium.
- the mesenchymal stem cells cultured in MS medium were comparable to the mesenchymal stem cells cultured in DMEM / F12 medium.
- Example 4 Cell culture 4
- hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SF medium, SP medium and MS medium for 3 days (37). °C, 5% CO 2 ) was carried out. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected.
- MCP-3 Cytokine Chemokic Protein-3
- VEGF Vascular Endotherial Growth Factor
- the supernatant of mesenchymal stem cells cultured in R medium had a higher content of GRO, MCP-3 and VEGF eotaxin than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field.
- the mesenchymal stem cells cultured in other serum-free mediums had a lower content of any factor than the mesenchymal stem cells cultured in DMEM / F12 medium.
- Example 5 Proliferation of adipose-derived mesenchymal stem cells 1
- the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline.
- Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells.
- a serum-free medium for mesenchymal stem cells (Rohto) was added to the above-mentioned cell precipitation, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container.
- adipose tissue-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SP medium, and MS medium, respectively (37 ° C., 5). % CO 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (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.
- Example 6 Proliferation of adipose-derived mesenchymal stem cells 2
- Human fat-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively.
- PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (Fig. 4).
- 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.
- Example 7 Proliferation of umbilical cord-derived mesenchymal stem cells 1
- the umbilical cord collected with the consent of the donor was washed with saline.
- Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells.
- a serum-free medium for mesenchymal stem cells (Rohto) was added to the precipitation of the above cells, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and umbilical cord tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container.
- the obtained umbilical cord-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and SP medium, MS medium and MesenCultm MSC Basic Medium (Human). Culturing (37 ° C., 5% CO 2 ) was carried out in (STEMCELL Technologies, hereinafter referred to as "MC medium"). PostPDL was determined by measuring the number of each cell after culturing for 4 days, 7 days and 11 days (Fig. 5).
- 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.
- Example 8 Proliferation of umbilical cord-derived mesenchymal stem cells 2
- Human umbilical cord-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively (37 ° C., 5% CO 2 ).
- PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days, 10 days and 13 days (Fig. 6).
- 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.
- Example 9 Proliferation of bone marrow-derived mesenchymal stem cells
- Human bone marrow-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Thermo Fisher Scientific) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium (37 ° C, 5% CO, respectively). 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 2, 7, 12, and 15 days (Fig. 7).
- 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.
- Example 10 Adhesion of adipose-derived mesenchymal stem cells
- Human fat cell-derived stem cells (ADSC) (LONZA, hereinafter referred to as "ADSC") were placed in a culture flask for adherent cells (Nunc EasYFlash Cell Culture Flashs, T25, cat: 156637, no coating agent used) at 2000 cells / cm 2 .
- the cells were seeded and cultured in R medium, MC medium and SP medium for 48 hours (37 ° C., 5% CO 2 ), and the cell adhesion state was observed under a microscope. Micrographs are shown in FIG. 8 (R medium), FIG. 9 (MC medium), and FIG. 10 (SP medium).
- the R medium adipocyte-derived stem cells were observed to adhere to the flask and elongated, but in other serum-free media, the elongated adipocyte-derived stem cells could not be observed. From the above, it was confirmed that the R medium has higher cell adhesion than other serum-free media.
- THP-1 human acute monocytic leukemia-derived cell line THP-1 (RIKEN BioResource Center, hereinafter referred to as "THP-1") was expanded and cultured according to the recommended protocol to prepare a frozen stock. On the 9th day from the dormant sowing of THP-1, the THP-1 suspension was collected, and the THP-1 suspension was liquidated with Phrol 12-Myristate 13-Actate (Fuji Film Wako Pure Chemical Industries, Ltd.) at a final concentration of 100. It was added so as to be nM.
- Phrol 12-Myristate 13-Actate Fruji Film Wako Pure Chemical Industries, Ltd.
- THP-1 suspension was inoculated and cultured (37 ° C., 5% CO 2 ) to induce the differentiation of THP-1.
- the cryopreserved cells are thawed so that the viable cell concentration becomes 8.96 ⁇ 10 4 cells / mL.
- 10% FBS-RPMI medium was added, and LPS-EB (InvivoGen) was further added to a final concentration of 1 ⁇ g / 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).
- the prepared cells were added into the Transwell insert of the well and co-cultured for 2 days.
- Two days after the start of co-culture total RNA of THP-1 was collected, and the mRNA expression levels of CCL2, TNF and PDGFB were measured by quantitative PCR. From the mRNA expression level obtained in each sample, the suppression rate was determined using the formula (1).
- the primers shown in Table 5 were used.
- the results are shown in FIGS. 11 (CCL2), 12 (TNF) and 13 (PDGFB). 100 ⁇ (1- (amount of mRNA of each sample) / (amount of mRNA of LPS (+))) ... Equation (1)
- the osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 17th revised Japanese Pharmacopoeia, and the osmotic pressure is the osmotic pressure measurement described in the Japanese Pharmacopoeia. The measurement was performed with reference to the method (freezing point descent method). As the standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution), the Japanese Pharmacopoeia physiological saline solution Otsuka Raw Food Injection (manufactured by Otsuka Pharmaceutical Factory) was used.
- All serum-free media had lower viscosities than DMEM / F12 media, but R medium had the lowest viscosity.
- the SP medium had the same osmotic pressure as the DMEM / F12 medium, but the other serum-free media had a lower osmotic pressure than the DMEM / F12 medium, and the R medium had the lowest osmotic pressure. .. From the above results, it was suggested that the R medium may have strong adhesiveness to cells while having low viscosity and low osmotic pressure.
- Example 12 Effect on T cells 1
- 24-well plates were thawed cryopreserved hUCMSC after culturing in R media or SF medium, and plated at 1.33 ⁇ 10 5 cells / well, were cultured for 15 hours or more.
- carboxyfluorescein succinimidyl ester (5- (and-6) -Carboxyfluorescein diacetate succinidiyl ester, hereinafter referred to as "CFSE”
- CFSE carboxyfluorescein succinimidyl ester
- PBMC blood mononuclear cells
- PBMCs blood mononuclear cells
- the subjects of analysis were CD4 positive and CD8 positive T cells, and these T cells were identified by antibody staining of CD3, CD4 and CD8.
- a gate was set at a position where the proliferation rate of the anti-CD3 / CD28 antibody-stimulated group was around 0.5%, and the same gate was used for the test group stimulated with anti-CD3 / CD28 antibody.
- hADMSC human adipose-derived mesenchymal cells
- Example 13 Effect on T cells 2
- hUCMSC cryopreserved was thawed and the proportion of T cells proliferated in the same manner as in Example 12 was measured.
- the proportion of proliferated CD4-positive T cells and CD8-positive T cells decreased in the group co-cultured with hUCMSC compared to the group not co-cultured with hUCMSC stimulated with anti-CD3 / CD28 antibody (CNTL). 16 and 17).
- mesenchymal stem cells having high cell adhesion and a high cell proliferation rate can be provided.
- the mesenchymal stem cells of the present invention have an effect of reducing the mRNA expression of macrophages CCL2, TNF and PDGFB and suppressing the proliferation of activated T cells, and exert a remarkable anti-inflammatory effect.
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Abstract
The purpose of the present invention is to provide mesenchymal stem cells that have exceptional cell adhesiveness, do not require serum medium, and have an adequate cell proliferation rate. The present invention is mesenchymal stem cells characterized in that the amount of granulocyte colony stimulating factor (G-CSF) produced is enhanced. The present invention also includes mesenchymal stem cells characterized in that the amount of at least one selected from the group consisting of eotaxin, fractalkine, GRO, MCP-3, and VEGF produced is enhanced.
Description
本発明は、間葉系幹細胞及び間葉系幹細胞用培地に関する。
The present invention relates to a mesenchymal stem cell and a medium for mesenchymal stem cells.
近年、人体の様々な部位の組織、細胞、受精卵等の細胞培養を行い、培養された細胞を再生医療等に使用することが実用化されている。その一つに間葉系幹細胞がある。間葉系幹細胞は、Ｆｒｉｅｄｅｎｓｔｅｉｎによって初めて骨髄から単離された多分化能を有する前駆細胞である（非特許文献１参照）。間葉系幹細胞は、骨髄、臍帯、脂肪等の様々な組織に存在することが明らかにされており、間葉系幹細胞移植は、様々な難治性疾患に対する新しい治療方法として、期待されている（特許文献１～２参照）。最近では、脂肪組織、胎盤、臍帯、卵膜等の間質細胞に同等の機能を有する細胞が存在することが知られている。従って、間葉系幹細胞を間質細胞（Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｒｏｍａｌ Ｃｅｌｌ）と称することもある。
In recent years, it has been put into practical use to culture cells, cells, fertilized eggs, etc. of various parts of the human body and use the cultured cells for regenerative medicine and the like. One of them is mesenchymal stem cells. Mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells are present in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new therapeutic method for various intractable diseases ( See Patent Documents 1 and 2). Recently, it is known that there are cells having the same function as stromal cells such as adipose tissue, placenta, umbilical cord, and fetal membrane. Therefore, mesenchymal stem cells may be referred to as stromal cells (Mesenchymal Cell).
間葉系幹細胞の培養には従来血清培地が用いられてきた。細胞培養において、血清は成長因子、接着因子、ホルモン、脂質及びミネラルの供給源として重要な因子である。一方で、血清培地には、ウシ胎児血清等の血清が用いられるが、動物血清に起因するウイルス、細菌等の混入の危険性を含んでいる。また、血清にはロット差があり、安定した効果を得られ難いという問題点もある。そのため、血清を適切な栄養成分及びホルモン成分で置き換え、動物血清使用の問題を回避できる無血清培地の開発が行われてきたが、細胞の増殖速度が血清培地を用いた場合よりも遅い等の問題があった。
Conventionally, a serum medium has been used for culturing mesenchymal stem cells. In cell culture, serum is an important source of growth factors, adhesion factors, hormones, lipids and minerals. On the other hand, although serum such as fetal bovine serum is used as the serum medium, there is a risk of contamination with viruses, bacteria, etc. caused by animal serum. In addition, there is a problem that it is difficult to obtain a stable effect due to lot differences in serum. Therefore, serum-free media have been developed that can replace the serum with appropriate nutritional and hormonal components to avoid the problem of animal serum use, but the cell growth rate is slower than when the serum medium is used. There was a problem.
また、間葉系幹細胞の培養を従来の無血清培地で行った場合、細胞の接着性が弱いため、コラーゲン、ラミニン、フィブロネクチン等のコーティング剤により培養容器の細胞接着面をコーティングする必要があり、手間と費用がかかった。
In addition, when the mesenchymal stem cells are cultured in a conventional serum-free medium, the cell adhesion is weak, so it is necessary to coat the cell adhesion surface of the culture vessel with a coating agent such as collagen, laminin, or fibronectin. It took time and money.
本発明は、上述のような状況の中、細胞接着性に優れ、血清培地を必要とすることなく、十分な細胞の増殖速度が得られる間葉系幹細胞を提供することを目的とする。
An object of the present invention is to provide mesenchymal stem cells which are excellent in cell adhesion and can obtain a sufficient cell proliferation rate without the need for a serum medium under the above-mentioned circumstances.
上記課題を解決するために鋭意研究した結果、本発明者らは、間葉系幹細胞（ｍｅｓｅｎｃｈｙｍａｌ ｓｔｅｍ（ｓｔｒｏｍａｌ） ｃｅｌｌ；ＭＳＣ）中の顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））の産生（Ｐｒｏｄｕｃｔｉｏｎ）量が亢進した細胞は、接着に必要な時間が短く、細胞の増殖速度が速いことを見出し、本発明を完成させた。本発明によれば、細胞の接着性に優れ、細胞の増殖速度が速い間葉系幹細胞を提供できる。すなわち本発明の要旨は、以下の通りである。
As a result of diligent research to solve the above problems, the present inventors have found that granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) in mesenchymal stem (stromal) cells (MSC). The present invention was completed by finding that cells in which the amount of production (Production) was enhanced had a short time required for adhesion and a high cell proliferation rate. According to the present invention, it is possible to provide mesenchymal stem cells having excellent cell adhesion and a high cell proliferation rate. That is, the gist of the present invention is as follows.
［１］顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））の産生（Ｐｒｏｄｕｃｔｉｏｎ）量が亢進していることを特徴とする、間葉系幹細胞。
［２］エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦからなる群より選択される少なくとも一つの産生量が亢進していることを特徴とする、間葉系幹細胞。
［３］脂肪組織由来、臍帯組織由来、もしくは骨髄組織由来である、［１］又は［２］に記載の間葉系幹細胞。
［４］［１］から［３］のいずれかに記載の間葉系幹細胞を誘導する細胞用培地。 [1] Mesenchymal stem cells, characterized in that the amount of production (Production) of a granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased.
[2] A mesenchymal stem cell characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced.
[3] The mesenchymal stem cell according to [1] or [2], which is derived from adipose tissue, umbilical cord tissue, or myeloid tissue.
[4] A cell medium for inducing mesenchymal stem cells according to any one of [1] to [3].
［２］エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦからなる群より選択される少なくとも一つの産生量が亢進していることを特徴とする、間葉系幹細胞。
［３］脂肪組織由来、臍帯組織由来、もしくは骨髄組織由来である、［１］又は［２］に記載の間葉系幹細胞。
［４］［１］から［３］のいずれかに記載の間葉系幹細胞を誘導する細胞用培地。 [1] Mesenchymal stem cells, characterized in that the amount of production (Production) of a granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased.
[2] A mesenchymal stem cell characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced.
[3] The mesenchymal stem cell according to [1] or [2], which is derived from adipose tissue, umbilical cord tissue, or myeloid tissue.
[4] A cell medium for inducing mesenchymal stem cells according to any one of [1] to [3].
本発明によると、細胞の接着性に優れ、細胞の増殖速度が速い間葉系幹細胞を提供することができる。本発明の間葉系幹細胞は、培養において血清を用いなくとも、接着性に優れ、容器を選ばず、早い速度で増殖することができる。また、本発明の間葉系幹細胞は、マクロファージのＣＣＬ２、ＴＮＦ及びＰＤＧＦＢのｍＲＮＡ発現を低下させ、また活性化したＴ細胞の増殖を抑制する作用も有し、顕著な抗炎症効果を奏する。
According to the present invention, it is possible to provide mesenchymal stem cells having excellent cell adhesion and a high cell proliferation rate. The mesenchymal stem cells of the present invention have excellent adhesiveness and can proliferate at a high rate in any container without using serum in culture. In addition, the mesenchymal stem cells of the present invention have an effect of reducing the mRNA expression of macrophages CCL2, TNF and PDGFB and suppressing the proliferation of activated T cells, and exert a remarkable anti-inflammatory effect.
以下、本発明の間葉系幹細胞について詳細に説明する。
Hereinafter, the mesenchymal stem cells of the present invention will be described in detail.
［間葉系幹細胞］
本発明の間葉系幹細胞は、顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））の産生量が亢進していることを特徴とする。また、本発明の間葉系幹細胞は、エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦからなる群より選択される少なくとも一つの産生量が亢進していることを特徴とする。 [Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are characterized in that the production amount of granulocyte colony stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased. In addition, the mesenchymal stem cells of the present invention are characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced. do.
本発明の間葉系幹細胞は、顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））の産生量が亢進していることを特徴とする。また、本発明の間葉系幹細胞は、エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦからなる群より選択される少なくとも一つの産生量が亢進していることを特徴とする。 [Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are characterized in that the production amount of granulocyte colony stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased. In addition, the mesenchymal stem cells of the present invention are characterized in that the production amount of at least one selected from the group consisting of EOtaxIN, Fractalkine, GRO, MCP-3 and VEGF is enhanced. do.
顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））とは、造血幹細胞を特異的に好中球（顆粒球）に分化させる増殖因子である。エオタキシン（ＥＯＴＡＸＩＮ）とは、好酸球に対して選択的に高い走化性を付与するＣＣケモカインの１つである。フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）とは、ケモカインと細胞接着分子の２つの活性を併せ持ち、活性化血管内皮細胞上に発現する細胞膜結合型ケモカインである。ＧＲＯとは、ＣＸＣＬ１としても知られ、分裂機能や好中球化学誘引物質であると考えられているケモカインである。ＭＣＰ－３（Ｍｏｎｏｃｙｔｅ Ｃｈｅｍｏｔａｃｔｉｃ Ｐｒｏｔｅｉｎ－３）とは、ｍｏｎｏｃｙｔｅ－ｓｐｅｃｉｆｉｃ ｃｙｔｏｋｉｎｅ－３とも呼ばれるＣＣケモカインであり、単球、マクロファージ等に対する走化因子である。血管内皮増殖因子（ＶＥＧＦ（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ））は血管新生を促すタンパク質であり、血管内皮細胞の増殖を始めとした血管新生過程の促進、血管透過性の亢進作用を有する。
Granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is a growth factor that specifically differentiates hematopoietic stem cells into neutrophils (granulocytes). EOTAXIN is one of CC chemokines that selectively imparts high chemotaxis to eosinophils. Fractalkine is a cell membrane-bound chemokine that has two activities, a chemokine and a cell adhesion molecule, and is expressed on activated vascular endothelial cells. GRO is a chemokine, also known as CXCL1, which is considered to be a mitotic function and neutrophil chemoattractant. MCP-3 (Monocyte Chemotactic Protein-3) is a CC chemokine also called cytokine-3, which is a chemotaxis factor for monocytes, macrophages and the like. Vascular Endothelial Growth Factor (VEGF) is a protein that promotes angiogenesis, and has an effect of promoting angiogenesis including proliferation of vascular endothelial cells and increasing vascular permeability.
本発明において、顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ）、エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３、ＶＥＧＦ等の各因子の産生量が亢進しているとは、細胞において各因子のｍＲＮＡ発現量が多いこと、各因子のタンパク質産生量が多いこと、各因子の分泌量が多いこと、又はいずれかの組み合わせであることを含む。また、本発明の間葉系幹細胞は、他の細胞に比べ、上記の各因子産生量が亢進されていればよいが、具体的には、本発明の間葉系幹細胞は、従来の培養条件下（例えば、１０％ＦＢＳ含有ＤＭＥＭ／Ｆ１２（Ｄｕｌｂｅｃｃｏ‘ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ Ｍｉｘｔｕｒｅ Ｆー１２）培地による培養）で得られる間葉系幹細胞に比べて、上記各因子の遺伝子発現量が多い、タンパク産生量が多い、又は各因子の分泌量が多ければよい。
In the present invention, it is said that the production amount of each factor such as granulocyte colony stimulating factor (G-CSF), eotaxin (EOTAXIN), fractalkine (FRACTALKINE), GRO, MCP-3, VEGF is increased in cells. In the above, the expression level of mRNA of each factor is high, the amount of protein produced by each factor is high, the amount of secretion of each factor is high, or any combination thereof is included. Further, the mesenchymal stem cell of the present invention may have an increased production amount of each of the above factors as compared with other cells. Specifically, the mesenchymal stem cell of the present invention has conventional culture conditions. Compared with the mesenchymal stem cells obtained below (for example, culture in DMEM / F12 (Dulvecco's Modified Eagle Medium: Nutrition Mixture F-12) medium containing 10% FBS), the gene expression level of each of the above factors is higher. It suffices if the amount of protein produced is large or the amount of each factor secreted is large.
上記各因子の産生量が亢進しているとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に比べ、ｍＲＮＡ発現量、タンパク産生量、分泌量が、１２０％以上、より好ましくは３００％以上、さらに好ましくは１５００％以上、特に好ましくは３０００％以上、最も好ましくは１００００％以上であることをいう。
The increase in the production amount of each of the above factors means that the mRNA expression level, protein production amount, and secretion amount are preferably 120% or more, more preferably 120% or more, as compared with the mesenchymal stem cells obtained under the conventional culture conditions. It means that it is 300% or more, more preferably 1500% or more, particularly preferably 3000% or more, and most preferably 10000% or more.
具体的には、Ｇ－ＣＳＦの産生量が亢進しているとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、５００％以上、より好ましくは１０００％以上、さらに好ましくは５０００％以上、特に好ましくは１５０００％以上であることをいう。
Specifically, it is said that the production amount of G-CSF is increased, preferably the production amount is 500% or more, more preferably 1000%, with respect to the mesenchymal stem cells obtained under the conventional culture conditions. Above, it means that it is more preferably 5000% or more, and particularly preferably 15000% or more.
エオタキシンの産生量が亢進しているあるとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、２００％以上、より好ましくは５００％以上、さらに好ましくは１０００％以上、特に好ましくは３０００％以上であることをいう。
The fact that the amount of eotaxin produced is increased means that the amount of mesenchymal stem cells produced is preferably 200% or more, more preferably 500% or more, still more preferably 1000, with respect to the mesenchymal stem cells obtained under conventional culture conditions. % Or more, particularly preferably 3000% or more.
フラクタルカインの産生量が亢進しているあるとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、１１０％以上、より好ましくは１２０％以上、さらに好ましくは１３０％以上、特に好ましくは１４０％以上であることをいう。
The fact that the amount of fractalkine produced is increased means that the amount of fractalkine produced is preferably 110% or more, more preferably 120% or more, still more preferably 120% or more, based on the mesenchymal stem cells obtained under conventional culture conditions. It means that it is 130% or more, particularly preferably 140% or more.
ＧＲＯの産生量が亢進しているとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、１２０％以上、より好ましくは３００％以上、さらに好ましくは５００％以上、特に好ましくは１５００％以上であることをいう。
The fact that the amount of GRO produced is increased means that the amount of GRO produced is preferably 120% or more, more preferably 300% or more, still more preferably 500%, based on the mesenchymal stem cells obtained under conventional culture conditions. The above is particularly preferably 1500% or more.
ＭＣＰ－３の産生量が亢進しているとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、１２０％以上、より好ましくは１５０％以上、さらに好ましくは２００％以上、特に好ましくは３００％以上であることをいう。
The increase in the production amount of MCP-3 means that the production amount is preferably 120% or more, more preferably 150% or more, still more preferably 150% or more, based on the mesenchymal stem cells obtained under the conventional culture conditions. It means that it is 200% or more, particularly preferably 300% or more.
ＶＥＧＦの産生量が亢進しているとは、好ましくは従来の培養条件下で得られる間葉系幹細胞に対し、その産生量が、１１０％以上、より好ましくは１２０％以上、さらに好ましくは１３０％以上、特に好ましくは１４０％以上であることをいう。
The fact that the amount of VEGF produced is increased means that the amount of VEGF produced is preferably 110% or more, more preferably 120% or more, still more preferably 130%, based on the mesenchymal stem cells obtained under conventional culture conditions. The above is particularly preferably 140% or more.
本発明において間葉系幹細胞とは、間葉系に属する一種以上の細胞（骨細胞、心筋細胞、軟骨細胞、腱細胞、脂肪細胞など）への分化能を有し、当該能力を維持したまま増殖できる細胞を意味する。本発明において用いる間葉系幹細胞なる用語は、間質細胞と同じ細胞を意味し、両者を特に区別するものではない。また、単に間葉系細胞と表記される場合もある。間葉系幹細胞を含む組織としては、例えば、脂肪組織、臍帯、骨髄、臍帯血、子宮内膜、胎盤、羊膜、絨毛膜、脱落膜、真皮、骨格筋、骨膜、歯小嚢、歯根膜、歯髄、歯胚等が挙げられる。例えば脂肪組織由来間葉系幹細胞とは、脂肪組織に含有される間葉系幹細胞を意味し、脂肪組織由来間質細胞と称してもよい。これらのうち、脂肪組織由来間葉系幹細胞、臍帯由来間葉系幹細胞、骨髄由来間葉系幹細胞、胎盤由来間葉系幹細胞、歯髄由来間葉系幹細胞が好ましく、脂肪組織由来間葉系幹細胞、臍帯由来間葉系幹細胞、骨髄由来間葉系幹細胞がより好ましい。
In the present invention, the mesenchymal stem cell has an ability to differentiate into one or more cells belonging to the mesenchymal system (osteocytes, myocardial cells, chondrocytes, tendon cells, adipocytes, etc.), and maintains the ability. It means a cell that can proliferate. The term mesenchymal stem cell used in the present invention means the same cell as a stromal cell, and does not particularly distinguish between the two. It may also be simply referred to as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, cord blood, endometrial membrane, placenta, amniotic membrane, chorion, decidua, dermatitis, skeletal muscle, bone membrane, tooth follicles, and periodontal ligament. Examples include dental pulp and tooth germ. For example, adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue, and may 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 preferable, and adipose tissue-derived mesenchymal stem cells, Umbilical band-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells are more preferable.
本発明における間葉系幹細胞の種として、ヒト、ウマ、ウシ、ヒツジ、ブタ、イヌ、ネコ、ラビット、マウス、ラットが挙げられる。
Examples of the species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats.
本発明において間葉系幹細胞とは、間葉系幹細胞を含む任意の細胞集団を意味する。当該細胞集団は、少なくとも２０％以上、好ましくは、３０％以上、４０％以上、５０％以上、６０％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、９３％以上、９６％以上、９７％以上、９８％以上又は９９％以上が間葉系幹細胞である。
In the present invention, the mesenchymal stem cell means an arbitrary cell population including the mesenchymal stem cell. The cell population is 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.
本発明において脂肪組織とは、脂肪細胞、及び微小血管細胞等を含む間質細胞を含有する組織を意味し、例えば、哺乳動物の皮下脂肪を外科的切除又は吸引して得られる組織である。
In the present invention, adipose tissue means a tissue containing stromal cells including adipocytes and microvascular cells, and is, for example, a tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal.
本発明において臍帯とは、胎児と胎盤を結ぶ白い管状の組織であり、臍帯静脈、臍帯動脈、膠様組織（ウォートンジェリー；Ｗｈａｒｔｏｎ’ｓ Ｊｅｌｌｙ）、臍帯基質自体等から構成され、間葉系幹細胞を多く含む。
In the present invention, the umbilical cord is a white tubular tissue that connects the fetal body and the placenta, and is composed of umbilical veins, umbilical arteries, glue-like tissues (Wharton's Jelly), umbilical cord matrix itself, and the like, and is composed of mesenchymal stem cells. Including a lot.
本発明において骨髄とは、骨の内腔を満たしている柔組織のことをいい、造血器官である。骨髄中には骨髄液が存在し、その中に存在する細胞を骨髄細胞と呼ぶ。骨髄細胞には、赤血球、顆粒球、巨核球、リンパ球、脂肪細胞等の他、間葉系幹細胞、造血幹細胞、血管内皮前駆細胞等が含まれている。骨髄細胞は、例えば、ヒト腸骨、長管骨、又はその他の骨から採取することができる。
In the present invention, the bone marrow is a parenchyma that fills the lumen of bone and is a hematopoietic organ. Bone marrow fluid exists in the bone marrow, and the cells existing in it are called bone marrow cells. Bone marrow cells include mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells and the like, as well as red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like. Bone marrow cells can be harvested from, for example, human ilium, long bone, or other bone.
（間葉系幹細胞の調製方法）
本発明の間葉系幹細胞の調製方法は特に限定されないが、例えば以下のようにして調製することができる。すなわち、脂肪組織、臍帯、骨髄等の組織から、当業者に公知の方法に従って、間葉系幹細胞を分離して、特定の培地を用いた培養により、各因子の産生量が亢進した間葉系幹細胞を誘導することで、本発明の間葉系幹細胞を取得することができる。この誘導によって得られる細胞集団において、細胞集団の５０％以上が本発明の細胞であることが好ましく、７０％以上が本発明の細胞であることがより好ましく、８０％以上が本発明の細胞であることがさらに好ましく、９０％以上が本発明の細胞であることが特に好ましく、実質的に本発明の細胞の均一な細胞集団であることが最も好ましい。 (Method of preparing mesenchymal stem cells)
The method for preparing mesenchymal stem cells of the present invention is not particularly limited, but can be prepared as follows, for example. That is, a mesenchymal system in which the production amount of each factor was enhanced by separating mesenchymal stem cells from tissues such as adipose tissue, umbilical cord, and bone marrow according to a method known to those skilled in the art and culturing them in a specific medium. By inducing stem cells, the mesenchymal stem cells of the present invention can be obtained. In the cell population obtained by this induction, 50% or more of the cell population is preferably the cells of the present invention, 70% or more is more preferably the cells of the present invention, and 80% or more is the cells of the present invention. More preferably, 90% or more of the cells of the present invention are particularly preferable, and substantially a uniform cell population of the cells of the present invention is most preferable.
本発明の間葉系幹細胞の調製方法は特に限定されないが、例えば以下のようにして調製することができる。すなわち、脂肪組織、臍帯、骨髄等の組織から、当業者に公知の方法に従って、間葉系幹細胞を分離して、特定の培地を用いた培養により、各因子の産生量が亢進した間葉系幹細胞を誘導することで、本発明の間葉系幹細胞を取得することができる。この誘導によって得られる細胞集団において、細胞集団の５０％以上が本発明の細胞であることが好ましく、７０％以上が本発明の細胞であることがより好ましく、８０％以上が本発明の細胞であることがさらに好ましく、９０％以上が本発明の細胞であることが特に好ましく、実質的に本発明の細胞の均一な細胞集団であることが最も好ましい。 (Method of preparing mesenchymal stem cells)
The method for preparing mesenchymal stem cells of the present invention is not particularly limited, but can be prepared as follows, for example. That is, a mesenchymal system in which the production amount of each factor was enhanced by separating mesenchymal stem cells from tissues such as adipose tissue, umbilical cord, and bone marrow according to a method known to those skilled in the art and culturing them in a specific medium. By inducing stem cells, the mesenchymal stem cells of the present invention can be obtained. In the cell population obtained by this induction, 50% or more of the cell population is preferably the cells of the present invention, 70% or more is more preferably the cells of the present invention, and 80% or more is the cells of the present invention. More preferably, 90% or more of the cells of the present invention are particularly preferable, and substantially a uniform cell population of the cells of the present invention is most preferable.
本発明の間葉系幹細胞を細胞医薬として用いるという観点から、血清等の異種由来成分を含まない（ゼノフリー）培地を用いることが好ましい。このような培地の中でも、間葉系幹細胞をＧ－ＣＳＦ等の特定の因子の産出量が亢進している本発明の間葉系幹細胞とする効果に優れる観点からは、粘度が１．００～１．２０の範囲であり、１．０５～１．１０であることが好ましい。また、培地の浸透圧は、１．００～１．１０の範囲であり、１．０４～１．０５であることが好ましい。間葉系幹細胞をＧ－ＣＳＦ等の特定の因子の産出量が亢進している本発明の間葉系幹細胞とするために有効である培地は、本発明の範囲である。
From the viewpoint of using the mesenchymal stem cells of the present invention as a cell medicine, it is preferable to use a medium containing no heterologous components such as serum (Xenofree). Even in such a medium, the mesenchymal stem cells have a viscosity of 1.00 to 1.00 to higher, from the viewpoint of excellent effect of converting the mesenchymal stem cells into the mesenchymal stem cells of the present invention in which the production amount of a specific factor such as G-CSF is enhanced. It is in the range of 1.20, preferably 1.05 to 1.10. The osmotic pressure of the medium is in the range of 1.00 to 1.10, preferably 1.04 to 1.05. The medium effective for converting the mesenchymal stem cells into the mesenchymal stem cells of the present invention in which the production of specific factors such as G-CSF is enhanced is within the scope of the present invention.
選択された細胞について、本発明における脂肪組織由来間葉系幹細胞であることを確認するために、表面抗原についてフローサイトメトリー等を用いて従来の方法で解析してもよい。さらに、各細胞系列に分化する能力について検査してもよく、このような分化は、従来の方法で行うことができる。
In order to confirm that the selected cells are adipose tissue-derived mesenchymal stem cells in the present invention, the surface antigen may be analyzed by a conventional method using flow cytometry or the like. In addition, the ability to differentiate into each cell lineage may be tested, and such differentiation can be performed by conventional methods.
本発明における間葉系幹細胞は、上述の通り調製することができるが、Ｇ－ＣＳＦ等の特定の因子の産出量が亢進すること以外に、さらに次の特性を持つ細胞として定義してもよい；
（１）標準培地での培養条件で、プラスチックに接着性を示す、
（２）表面抗原ＣＤ４４、ＣＤ７３、ＣＤ９０が陽性であり、ＣＤ３１、ＣＤ４５が陰性である、
（３）培養条件にて骨細胞、脂肪細胞、軟骨細胞に分化可能である。 The mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics in addition to increasing the production of specific factors such as G-CSF. ;
(1) Shows adhesiveness to plastic under culture conditions in standard medium.
(2) The surface antigens CD44, CD73 and CD90 are positive, and CD31 and CD45 are negative.
(3) It can be differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.
（１）標準培地での培養条件で、プラスチックに接着性を示す、
（２）表面抗原ＣＤ４４、ＣＤ７３、ＣＤ９０が陽性であり、ＣＤ３１、ＣＤ４５が陰性である、
（３）培養条件にて骨細胞、脂肪細胞、軟骨細胞に分化可能である。 The mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics in addition to increasing the production of specific factors such as G-CSF. ;
(1) Shows adhesiveness to plastic under culture conditions in standard medium.
(2) The surface antigens CD44, CD73 and CD90 are positive, and CD31 and CD45 are negative.
(3) It can be differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.
本発明の間葉系幹細胞は、いずれの状態の細胞であってもよいが、例えば培養中の細胞を剥離して回収された細胞でもよいし、凍結保存液中に凍結された状態の細胞でもよい。
The mesenchymal stem cell of the present invention may be a cell in any state, for example, a cell recovered by exfoliating a cell in culture, or a cell frozen in a cryopreservation solution. good.
以下に、実施例及び試験例を挙げて本発明を詳細に説明するが、本発明はこれらの実施例等によって限定されるものではない。
Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples and the like.
［実施例１：細胞の培養１］
ヒト臍帯由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社、以下「ｈＵＣＭＳＣ」と言う）を、接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれ１０％ＦＢＳ ＤＭＥＭ／Ｆ１２培地（Ｓｉｇｍａ社、以下「ＤＭＥＭ／Ｆ１２培地」と言う）、Ｒ：Ｓｔｅｍ間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社、以下「Ｒ培地」と言う）、ＳｔｅｍＦｉｔ Ｆｏｒ Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌ（味の素社、以下「ＳＦ培地」と言う）及びＳＴＥＭＰＲＯ（登録商標） ＭＳＣ ＳＦＭ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社、以下「ＳＰ培地」と言う）で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中の顆粒球コロニー形成刺激因子（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ、以下「Ｇ－ＣＳＦ」と言う）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のＧ－ＣＳＦ含有量を１００％とした場合の各培地で培養した細胞の上清中のＧ－ＣＳＦ量を求めた。結果を表１に示した。 [Example 1: Cell culture 1]
Human umbilical mesenchymal stem cells (Promo Cell, hereinafter referred to as "hUCMSC") were seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and each was seeded with 10% FBS DMEM / F12 medium (Sigma). , Hereinafter referred to as "DMEM / F12 medium"), R: Stem mesenchymal stem cell serum-free medium (Rohto, hereinafter referred to as "R medium"), StemFit For Mesenchymal Stem Cell (Ajinomoto, hereinafter referred to as "SF medium") ) And STEMPRO® MSC SFM (Thermo Fisher Scientific, hereinafter referred to as "SP medium") were cultured for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using a MAGPIX® system (Merck Millipor) using a Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and granulocytes in cell supernatant. Granulocyte colony stimulating factor (hereinafter referred to as "G-CSF") was measured. The amount of G-CSF in the supernatant of the cells cultured in each medium was determined when the G-CSF content in the supernatant of the cells cultured in DMEM / F12 medium was 100%. The results are shown in Table 1.
ヒト臍帯由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社、以下「ｈＵＣＭＳＣ」と言う）を、接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれ１０％ＦＢＳ ＤＭＥＭ／Ｆ１２培地（Ｓｉｇｍａ社、以下「ＤＭＥＭ／Ｆ１２培地」と言う）、Ｒ：Ｓｔｅｍ間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社、以下「Ｒ培地」と言う）、ＳｔｅｍＦｉｔ Ｆｏｒ Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌ（味の素社、以下「ＳＦ培地」と言う）及びＳＴＥＭＰＲＯ（登録商標） ＭＳＣ ＳＦＭ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社、以下「ＳＰ培地」と言う）で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中の顆粒球コロニー形成刺激因子（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ、以下「Ｇ－ＣＳＦ」と言う）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のＧ－ＣＳＦ含有量を１００％とした場合の各培地で培養した細胞の上清中のＧ－ＣＳＦ量を求めた。結果を表１に示した。 [Example 1: Cell culture 1]
Human umbilical mesenchymal stem cells (Promo Cell, hereinafter referred to as "hUCMSC") were seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and each was seeded with 10% FBS DMEM / F12 medium (Sigma). , Hereinafter referred to as "DMEM / F12 medium"), R: Stem mesenchymal stem cell serum-free medium (Rohto, hereinafter referred to as "R medium"), StemFit For Mesenchymal Stem Cell (Ajinomoto, hereinafter referred to as "SF medium") ) And STEMPRO® MSC SFM (Thermo Fisher Scientific, hereinafter referred to as "SP medium") were cultured for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using a MAGPIX® system (Merck Millipor) using a Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and granulocytes in cell supernatant. Granulocyte colony stimulating factor (hereinafter referred to as "G-CSF") was measured. The amount of G-CSF in the supernatant of the cells cultured in each medium was determined when the G-CSF content in the supernatant of the cells cultured in DMEM / F12 medium was 100%. The results are shown in Table 1.
また、ｈＵＣＭＳＣを、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地およびＳＰ培地に播種し、細胞が静止（接着）するまでの時間をライブセルイメージングシステム（ＳＩ８０００、ＳＯＮＹ社）を用いて測定した。結果を表１に示した。
Further, hUCMSC was inoculated into DMEM / F12 medium, R medium, SF medium and SP medium, respectively, and the time until the cells became stationary (adhered) was measured using a live cell imaging system (SI8000, SONY). The results are shown in Table 1.
さらに、ｈＵＣＭＳＣを、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地およびＳＰ培地に播種後、２日間培養（３７℃、５％ＣＯ２）して細胞増殖活性をＷＳＴ－８（Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ－８）にて評価した（表１）。
Further, hUCMSC is inoculated into DMEM / F12 medium, R medium, SF medium and SP medium, respectively, and then cultured for 2 days (37 ° C., 5% CO 2 ) to increase the cell proliferation activity of WST-8 (Cell Counting Kit-8). ) (Table 1).
表１に示すとおり、Ｒ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と比較して、Ｇ－ＣＳＦ含有が高いことがわかった。これに対して他の無血清培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と比較して、Ｇ－ＣＳＦ含有が低いことがわかった。
As shown in Table 1, it was found that the supernatant of the mesenchymal stem cells cultured in R medium had a higher G-CSF content than the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium. In contrast, the supernatant of mesenchymal stem cells cultured in other serum-free medium was found to have a lower G-CSF content than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. ..
また、Ｒ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して培養容器への接着するまでの時間が短かった。これに対して他の無血清培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して、培養容器へ接着するまでの時間が長かった。
In addition, the mesenchymal stem cells cultured in R medium had a shorter time to adhere to the culture vessel than the mesenchymal stem cells cultured in DMEM / F12 medium. On the other hand, the mesenchymal stem cells cultured in other serum-free medium took a longer time to adhere to the culture vessel than the mesenchymal stem cells cultured in DMEM / F12 medium.
さらに、Ｒ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して細胞増殖活性が高かった。これに対して他の無血清培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して、細胞増殖活性が低かった。
Furthermore, the mesenchymal stem cells cultured in R medium had higher cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium. On the other hand, the mesenchymal stem cells cultured in other serum-free mediums had lower cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium.
以上の結果から、Ｒ培地で培養した間葉系幹細胞はＧ－ＣＳＦの分泌量が多く、また、細胞接着に要する時間が短く、細胞増殖活性が高いことが明らかとなった。
From the above results, it was clarified that the mesenchymal stem cells cultured in R medium secreted a large amount of G-CSF, took a short time for cell adhesion, and had high cell proliferation activity.
また、上記の培養に用いた培地が異なる細胞について、細胞接着に要する時間と、Ｇ－ＣＳＦの分泌量との関係、及び細胞増殖活性と、Ｇ－ＣＳＦの分泌量との関係をそれぞれ図１及び図２に示した。Ｇ－ＣＳＦの分泌量が多い細胞ほど、細胞接着に要する時間が短かった（図１）。Ｇ－ＣＳＦの分泌量が多い細胞ほど細胞増殖活性が高かった（図２）。
Further, for cells using different media used for the above culture, the relationship between the time required for cell adhesion and the amount of G-CSF secreted, and the relationship between the cell proliferation activity and the amount of G-CSF secreted are shown in FIG. And shown in FIG. The larger the amount of G-CSF secreted, the shorter the time required for cell adhesion (Fig. 1). The cells with higher G-CSF secretion had higher cell proliferation activity (Fig. 2).
［実施例２：細胞の培養２］
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のエオタキシン（Ｅｏｔａｘｉｎ）及びフラクタルカイン（Ｆｒａｃｔａｌｋｉｎｅ）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン含有量を１００％とした場合の各培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン量を求めた（表２）。 [Example 2: Cell culture 2]
In the same manner as in Example 1, hUCMSC was seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium and SP medium for 3 days (37 ° C., 5% CO 2), respectively. ) Was performed. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using the MAGPIX® system (Merck Millipore) using the Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. (Eotaxin) and Fractalkine were measured. The amounts of eotaxin and fractalkine in the supernatant of the cells cultured in each medium were determined when the content of eotaxin and fractalkine in the supernatant of the cells cultured in DMEM / F12 medium was 100% (Table 2). ).
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のエオタキシン（Ｅｏｔａｘｉｎ）及びフラクタルカイン（Ｆｒａｃｔａｌｋｉｎｅ）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン含有量を１００％とした場合の各培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン量を求めた（表２）。 [Example 2: Cell culture 2]
In the same manner as in Example 1, hUCMSC was seeded in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium and SP medium for 3 days (37 ° C., 5% CO 2), respectively. ) Was performed. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using the MAGPIX® system (Merck Millipore) using the Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. (Eotaxin) and Fractalkine were measured. The amounts of eotaxin and fractalkine in the supernatant of the cells cultured in each medium were determined when the content of eotaxin and fractalkine in the supernatant of the cells cultured in DMEM / F12 medium was 100% (Table 2). ).
また、ｈＵＣＭＳＣを、それぞれＤＭＥＭ／Ｆ２培地、Ｒ培地およびＳＰ培地に播種後、ライブセルイメージングシステム（ＳＩ８０００、ＳＯＮＹ社）を用いて、遊走距離を測定した（表２）。
In addition, hUCMSC was inoculated into DMEM / F2 medium, R medium and SP medium, respectively, and then the migration distance was measured using a live cell imaging system (SI8000, SONY) (Table 2).
以上の結果から、Ｒ培地で培養した間葉系幹細胞はエオタキシン及びフラクタルカインの分泌量が多く、また、遊走距離が長いことが明らかとなった。
From the above results, it was clarified that the mesenchymal stem cells cultured in R medium secreted a large amount of eotaxin and fractalkine and had a long migration distance.
表２に示すとおり、Ｒ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と比較して、エオタキシン及びフラクタルカインの含有が高いことがわかった。これに対してＳＰ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と同程度であった。
また、Ｒ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して有意に遊走距離が長かった。 As shown in Table 2, it was found that the supernatant of mesenchymal stem cells cultured in R medium contained a higher content of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field. On the other hand, the supernatant of the mesenchymal stem cells cultured in SP medium was similar to the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium.
In addition, the mesenchymal stem cells cultured in R medium had a significantly longer migration distance than the mesenchymal stem cells cultured in DMEM / F12 medium.
また、Ｒ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して有意に遊走距離が長かった。 As shown in Table 2, it was found that the supernatant of mesenchymal stem cells cultured in R medium contained a higher content of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field. On the other hand, the supernatant of the mesenchymal stem cells cultured in SP medium was similar to the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium.
In addition, the mesenchymal stem cells cultured in R medium had a significantly longer migration distance than the mesenchymal stem cells cultured in DMEM / F12 medium.
［実施例３：細胞の培養３］
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＭｅｓｅｎＰＲＯ ＲＳ（商標） Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社、以下「ＭＰ培地」と言う）で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のエオタキシン（Ｅｏｔａｘｉｎ）及びフラクタルカイン（Ｆｒａｃｔａｌｋｉｎｅ）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン含有量を１００％とした場合の各培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン量を求めた（表３）。 [Example 3: Cell culture 3]
In the same manner as in Example 1, hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and MesenPRO RS ™ Medium (Thermo Fisher Scientific), hereinafter ". It was cultured in MP medium) for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using the MAGPIX® system (Merck Millipore) using the Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. (Eotaxin) and Fractalkine were measured. The amounts of eotaxin and fractalkine in the supernatant of the cells cultured in each medium were determined when the content of eotaxin and fractalkine in the supernatant of the cells cultured in DMEM / F12 medium was 100% (Table 3). ).
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＭｅｓｅｎＰＲＯ ＲＳ（商標） Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社、以下「ＭＰ培地」と言う）で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のエオタキシン（Ｅｏｔａｘｉｎ）及びフラクタルカイン（Ｆｒａｃｔａｌｋｉｎｅ）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン含有量を１００％とした場合の各培地で培養した細胞の上清中のエオタキシン及びフラクタルカイン量を求めた（表３）。 [Example 3: Cell culture 3]
In the same manner as in Example 1, hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and MesenPRO RS ™ Medium (Thermo Fisher Scientific), hereinafter ". It was cultured in MP medium) for 3 days (37 ° C., 5% CO 2 ). Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using the MAGPIX® system (Merck Millipore) using the Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. (Eotaxin) and Fractalkine were measured. The amounts of eotaxin and fractalkine in the supernatant of the cells cultured in each medium were determined when the content of eotaxin and fractalkine in the supernatant of the cells cultured in DMEM / F12 medium was 100% (Table 3). ).
また、ｈＵＣＭＳＣを、それぞれＤＭＥＭ／Ｆ２培地、Ｒ培地及びＭＰ培地に播種後、ライブセルイメージングシステム（ＳＩ８０００、ＳＯＮＹ社）を用いて、細胞が静止（接着）するまでの時間及び遊走距離を測定した（表３）。
In addition, after seeding hUCMSC in DMEM / F2 medium, R medium and MP medium, respectively, the time until the cells rested (adhered) and the migration distance were measured using a live cell imaging system (SI8000, SONY). (Table 3).
さら、ｈＵＣＭＳＣを、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＭＰ培地に播種後、２日間培養（３７℃、５％ＣＯ２）して細胞増殖活性をＷＳＴ－８（Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ－８）にて評価した（表３）。
Furthermore, hUCMSC was seeded in DMEM / F12 medium, R medium and MP medium, respectively, and then cultured for 2 days (37 ° C., 5% CO 2 ) to increase cell proliferation activity in WST-8 (Cell Counting Kit-8). Evaluated (Table 3).
表３に示すとおり、Ｒ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と比較して、エオタキシン及びフラクタルカインの含有が高いことがわかった。これに対してＭＳ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と同程度であった。
As shown in Table 3, it was found that the supernatant of mesenchymal stem cells cultured in R medium contained a higher content of eotaxin and fractalkine than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field. On the other hand, the supernatant of the mesenchymal stem cells cultured in MS medium was similar to the supernatant of the mesenchymal stem cells cultured in DMEM / F12 medium.
また、Ｒ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して、容器への接着までの時間が有意に短く、細胞増殖活性が高かった。これに対してＭＳ培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と同程度であった。
In addition, the mesenchymal stem cells cultured in R medium had a significantly shorter time to adhere to the container and higher cell proliferation activity than the mesenchymal stem cells cultured in DMEM / F12 medium. On the other hand, the mesenchymal stem cells cultured in MS medium were comparable to the mesenchymal stem cells cultured in DMEM / F12 medium.
以上の結果から、Ｒ培地で培養した間葉系幹細胞はエオタキシン及びフラクタルカインの分泌量が多く、また遊走距離が長く、細胞増殖活性が高いことが明らかとなった。
From the above results, it was clarified that the mesenchymal stem cells cultured in R medium secreted a large amount of eotaxin and fractalkine, had a long migration distance, and had high cell proliferation activity.
［実施例４：細胞の培養４］
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＳＰ培地及びＭＳ培地で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のＧＲＯ、Ｍｏｎｏｃｙｔｅ Ｃｈｅｍｏｔａｃｔｉｃ Ｐｒｏｔｅｉｎ－３（以下「ＭＣＰ－３」と言う）及び血管内皮増殖因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ、以下「ＶＥＧＦ」と言う）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中の各成分の含有量を１００％とした場合の各培地で培養した細胞の上清中の各成分の含有量を求めた（表４）。 [Example 4: Cell culture 4]
In the same manner as in Example 1, hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SF medium, SP medium and MS medium for 3 days (37). ℃, 5% CO 2 ) was carried out. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using a MAGPIX® system (Merck Millipor) using a Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. , Cytokine Chemokic Protein-3 (hereinafter referred to as "MCP-3") and vascular endothelial growth factor (Vascular Endotherial Growth Factor, hereinafter referred to as "VEGF") were measured. The content of each component in the supernatant of cells cultured in each medium was determined when the content of each component in the supernatant of cells cultured in DMEM / F12 medium was 100% (Table 4). ..
実施例１と同様に、ｈＵＣＭＳＣを接着細胞用Ｔ７５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＳＰ培地及びＭＳ培地で３日間培養（３７℃、５％ＣＯ２）を行った。その後、細胞剥離液を用いて継代し、さらに４日間培養（３７℃、５％ＣＯ２）後の上清を回収した。Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ／Ｃｈｅｍｏｋｉｎｅ Ｍａｇｎｅｔｉｃ Ｂｅａｄ Ｐａｎｅｌ （ＥＭＤ Ｍｉｌｌｉｐｏｒｅ社、ＣＡＴ．：ＨＣＹＴＯＭＡＧ－６０Ｋ）を用いてＭＡＧＰＩＸ（登録商標）システム（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒ社）を使用して、マルチプレックスアッセイを行い、細胞上清中のＧＲＯ、Ｍｏｎｏｃｙｔｅ Ｃｈｅｍｏｔａｃｔｉｃ Ｐｒｏｔｅｉｎ－３（以下「ＭＣＰ－３」と言う）及び血管内皮増殖因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ、以下「ＶＥＧＦ」と言う）を測定した。なお、ＤＭＥＭ／Ｆ１２培地で培養した細胞の上清中の各成分の含有量を１００％とした場合の各培地で培養した細胞の上清中の各成分の含有量を求めた（表４）。 [Example 4: Cell culture 4]
In the same manner as in Example 1, hUCMSC was inoculated in a T75 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SF medium, SP medium and MS medium for 3 days (37). ℃, 5% CO 2 ) was carried out. Then, the cells were subcultured using a cell exfoliating solution, and the supernatant after further culturing for 4 days (37 ° C., 5% CO 2 ) was collected. Multiplex assay was performed using a MAGPIX® system (Merck Millipor) using a Human Cytokine / Chemokine Magnetic Bed Panel (EMD Millipore, CAT .: HCYTOMAG-60K) and in the cell supernatant. , Cytokine Chemokic Protein-3 (hereinafter referred to as "MCP-3") and vascular endothelial growth factor (Vascular Endotherial Growth Factor, hereinafter referred to as "VEGF") were measured. The content of each component in the supernatant of cells cultured in each medium was determined when the content of each component in the supernatant of cells cultured in DMEM / F12 medium was 100% (Table 4). ..
表４に示すとおり、Ｒ培地で培養した間葉系幹細胞の上清は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞の上清と比較して、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦエオタキシン含有が高かった。これに対して他の無血清培地で培養した間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した間葉系幹細胞と比較して、いずれの因子の含有量も低かった。
As shown in Table 4, the supernatant of mesenchymal stem cells cultured in R medium had a higher content of GRO, MCP-3 and VEGF eotaxin than the supernatant of mesenchymal stem cells cultured in DMEM / F12 medium. rice field. In contrast, the mesenchymal stem cells cultured in other serum-free mediums had a lower content of any factor than the mesenchymal stem cells cultured in DMEM / F12 medium.
［実施例５：脂肪由来間葉系幹細胞の増殖１］
ヒトドナーから同意を得た後、脂肪吸引法で得た皮下脂肪組織を生理食塩液で洗浄した。細胞外基質の破壊、及び細胞の単離を達成するために、コラゲナーゼ（Ｒｏｃｈｅ ｄｉａｇｎｏｓｔｉｃｓ社）（溶媒は生理食塩液）を添加し、３７℃で９０分間振倒し、分散した。続いて、この上記懸濁液を８００ｇで５分間、遠心分離して間質血管細胞群の沈殿を得た。上記細胞の沈殿に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）を加え、当該細胞懸濁液を４００ｇで５分間遠心分離し、上清除去後に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）に再懸濁し、フラスコに細胞を播種した。細胞を３７℃で数日間、５％ＣＯ２中で培養した。数日後に培養物をＰＢＳで洗浄して、培養液中に含まれていた血球や脂肪組織の残存等を除去し、プラスチック容器に接着している間葉系幹細胞を得た。得られた脂肪組織由来間葉系幹細胞を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地及びＭＳ培地で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図３）。 [Example 5: Proliferation of adipose-derived mesenchymal stem cells 1]
After obtaining the consent of the human donor, the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline. Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the above-mentioned cell precipitation, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container. The obtained adipose tissue-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SP medium, and MS medium, respectively (37 ° C., 5). % CO 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (Fig. 3).
ヒトドナーから同意を得た後、脂肪吸引法で得た皮下脂肪組織を生理食塩液で洗浄した。細胞外基質の破壊、及び細胞の単離を達成するために、コラゲナーゼ（Ｒｏｃｈｅ ｄｉａｇｎｏｓｔｉｃｓ社）（溶媒は生理食塩液）を添加し、３７℃で９０分間振倒し、分散した。続いて、この上記懸濁液を８００ｇで５分間、遠心分離して間質血管細胞群の沈殿を得た。上記細胞の沈殿に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）を加え、当該細胞懸濁液を４００ｇで５分間遠心分離し、上清除去後に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）に再懸濁し、フラスコに細胞を播種した。細胞を３７℃で数日間、５％ＣＯ２中で培養した。数日後に培養物をＰＢＳで洗浄して、培養液中に含まれていた血球や脂肪組織の残存等を除去し、プラスチック容器に接着している間葉系幹細胞を得た。得られた脂肪組織由来間葉系幹細胞を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地及びＭＳ培地で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図３）。 [Example 5: Proliferation of adipose-derived mesenchymal stem cells 1]
After obtaining the consent of the human donor, the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline. Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the above-mentioned cell precipitation, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container. The obtained adipose tissue-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium, R medium, SP medium, and MS medium, respectively (37 ° C., 5). % CO 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (Fig. 3).
図３に示すとおり、Ｒ培地で培養した脂肪由来間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地及びＳＰ培地で培養した脂肪由来間葉系幹細胞と比較して、細胞増殖活性が高かった。
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.
［実施例６：脂肪由来間葉系幹細胞の増殖２］
ヒト脂肪由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図４）。 [Example 6: Proliferation of adipose-derived mesenchymal stem cells 2]
Human fat-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (Fig. 4).
ヒト脂肪由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図４）。 [Example 6: Proliferation of adipose-derived mesenchymal stem cells 2]
Human fat-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days and 11 days (Fig. 4).
実施例５と同様、Ｒ培地で培養した脂肪由来間葉系幹細胞は、ＤＭＥＭ／Ｆ１２で培養した脂肪由来間葉系幹細胞と比較して、細胞増殖活性が高かった。
Similar to 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.
［実施例７：臍帯由来間葉系幹細胞の増殖１］
提供者の同意を得て採取された臍帯を生理食塩液で洗浄した。細胞外基質の破壊、及び細胞の単離を達成するために、コラゲナーゼ（Ｒｏｃｈｅ ｄｉａｇｎｏｓｔｉｃｓ社）（溶媒は生理食塩液）を添加し、３７℃で９０分間振倒し、分散した。続いて、この上記懸濁液を８００ｇで５分間、遠心分離して間質血管細胞群の沈殿を得た。上記細胞の沈殿に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）を加え、当該細胞懸濁液を４００ｇで５分間遠心分離し、上清除去後に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）に再懸濁し、フラスコに細胞を播種した。細胞を３７℃で数日間、５％ＣＯ２中で培養した。数日後に培養物をＰＢＳで洗浄して、培養液中に含まれていた血球や臍帯組織の残存等を除去し、プラスチック容器に接着している間葉系幹細胞を得た。得られた臍帯由来間葉系幹細胞を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地、ＭＳ培地及びＭｅｓｅｎＣｕｌｔｔｍ ＭＳＣ Ｂａｓａｌ Ｍｅｄｉｕｍ（Ｈｕｍａｎ）（ＳＴＥＭＣＥＬＬ Ｔｅｃｈｎｏｌｏｇｉｅｓ社、以下「ＭＣ培地」と言う）で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図５）。 [Example 7: Proliferation of umbilical cord-derived mesenchymal stem cells 1]
The umbilical cord collected with the consent of the donor was washed with saline. Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the precipitation of the above cells, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and umbilical cord tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container. The obtained umbilical cord-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and SP medium, MS medium and MesenCultm MSC Basic Medium (Human). Culturing (37 ° C., 5% CO 2 ) was carried out in (STEMCELL Technologies, hereinafter referred to as "MC medium"). PostPDL was determined by measuring the number of each cell after culturing for 4 days, 7 days and 11 days (Fig. 5).
提供者の同意を得て採取された臍帯を生理食塩液で洗浄した。細胞外基質の破壊、及び細胞の単離を達成するために、コラゲナーゼ（Ｒｏｃｈｅ ｄｉａｇｎｏｓｔｉｃｓ社）（溶媒は生理食塩液）を添加し、３７℃で９０分間振倒し、分散した。続いて、この上記懸濁液を８００ｇで５分間、遠心分離して間質血管細胞群の沈殿を得た。上記細胞の沈殿に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）を加え、当該細胞懸濁液を４００ｇで５分間遠心分離し、上清除去後に間葉系幹細胞用無血清培地（Ｒｏｈｔｏ社）に再懸濁し、フラスコに細胞を播種した。細胞を３７℃で数日間、５％ＣＯ２中で培養した。数日後に培養物をＰＢＳで洗浄して、培養液中に含まれていた血球や臍帯組織の残存等を除去し、プラスチック容器に接着している間葉系幹細胞を得た。得られた臍帯由来間葉系幹細胞を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地及びＳＰ培地、ＭＳ培地及びＭｅｓｅｎＣｕｌｔｔｍ ＭＳＣ Ｂａｓａｌ Ｍｅｄｉｕｍ（Ｈｕｍａｎ）（ＳＴＥＭＣＥＬＬ Ｔｅｃｈｎｏｌｏｇｉｅｓ社、以下「ＭＣ培地」と言う）で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間及び１１日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図５）。 [Example 7: Proliferation of umbilical cord-derived mesenchymal stem cells 1]
The umbilical cord collected with the consent of the donor was washed with saline. Collagenase (Roche diagnostics) (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the precipitation of the above cells, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. for several days in 5% CO 2 . After a few days, the culture was washed with PBS to remove residual blood cells and umbilical cord tissue contained in the culture medium to obtain mesenchymal stem cells adhering to a plastic container. The obtained umbilical cord-derived mesenchymal stem cells were seeded in a T25 flask for adherent cells (Corning) at 5000 cells / cm 2 , respectively, and DMEM / F12 medium, R medium and SP medium, MS medium and MesenCultm MSC Basic Medium (Human). Culturing (37 ° C., 5% CO 2 ) was carried out in (STEMCELL Technologies, hereinafter referred to as "MC medium"). PostPDL was determined by measuring the number of each cell after culturing for 4 days, 7 days and 11 days (Fig. 5).
Ｒ培地で培養した臍帯由来間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地及び他の無血清培地で培養した臍帯由来間葉系幹細胞と比較して、細胞増殖活性が高かった。
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.
［実施例８：臍帯由来間葉系幹細胞の増殖２］
ヒト臍帯由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間、１０日間及び１３日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図６）。 [Example 8: Proliferation of umbilical cord-derived mesenchymal stem cells 2]
Human umbilical cord-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively (37 ° C., 5% CO 2 ). Was done. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days, 10 days and 13 days (Fig. 6).
ヒト臍帯由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｃｏｒｎｉｎｇ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養（３７℃、５％ＣＯ２）を行った。４日間、７日間、１０日間及び１３日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図６）。 [Example 8: Proliferation of umbilical cord-derived mesenchymal stem cells 2]
Human umbilical cord-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Corning) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium, respectively (37 ° C., 5% CO 2 ). Was done. PostPDL was determined by measuring the number of cells after culturing for 4 days, 7 days, 10 days and 13 days (Fig. 6).
Ｒ培地で培養した臍帯由来間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した臍帯由来間葉系幹細胞と比較して、細胞増殖活性が高かった。
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.
［実施例９：骨髄由来間葉系幹細胞の増殖］
ヒト骨髄由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養（３７℃、５％ＣＯ２）を行った。２日間、７日間、１２日間及び１５日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図７）。 [Example 9: Proliferation of bone marrow-derived mesenchymal stem cells]
Human bone marrow-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Thermo Fisher Scientific) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium (37 ° C, 5% CO, respectively). 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 2, 7, 12, and 15 days (Fig. 7).
ヒト骨髄由来間葉系幹細胞（Ｐｒｏｍｏ Ｃｅｌｌ社）を接着細胞用Ｔ２５フラスコ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）に５０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＤＭＥＭ／Ｆ１２培地及びＲ培地で培養（３７℃、５％ＣＯ２）を行った。２日間、７日間、１２日間及び１５日間培養後の各細胞数を測定してＰｏｓｔＰＤＬを求めた（図７）。 [Example 9: Proliferation of bone marrow-derived mesenchymal stem cells]
Human bone marrow-derived mesenchymal stem cells (Promo Cell) were seeded in T25 flasks for adherent cells (Thermo Fisher Scientific) at 5000 cells / cm 2 , and cultured in DMEM / F12 medium and R medium (37 ° C, 5% CO, respectively). 2 ) was performed. PostPDL was determined by measuring the number of cells after culturing for 2, 7, 12, and 15 days (Fig. 7).
Ｒ培地で培養した臍帯由来間葉系幹細胞は、ＤＭＥＭ／Ｆ１２培地で培養した臍帯由来間葉系幹細胞と比較して、細胞増殖活性が高かった。
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.
［実施例１０：脂肪由来間葉系幹細胞の接着］
ヒト脂肪細胞由来幹細胞（ＡＤＳＣ）（ＬＯＮＺＡ社、以下「ＡＤＳＣ」と言う）を、接着細胞用培養フラスコ（Ｎｕｎｃ ＥａｓＹＦｌａｓｋ Ｃｅｌｌ Ｃｕｌｔｕｒｅ Ｆｌａｓｋｓ、Ｔ２５、ｃａｔ：１５６３６７、コーティング剤未使用）に２０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＲ培地、ＭＣ培地及びＳＰ培地で４８時間培養（３７℃、５％ＣＯ２）して細胞の接着状態を顕微鏡で観察した。顕微鏡写真を図８（Ｒ培地）、図９（ＭＣ培地）、図１０（ＳＰ培地）に示す。 [Example 10: Adhesion of adipose-derived mesenchymal stem cells]
Human fat cell-derived stem cells (ADSC) (LONZA, hereinafter referred to as "ADSC") were placed in a culture flask for adherent cells (Nunc EasYFlash Cell Culture Flashs, T25, cat: 156637, no coating agent used) at 2000 cells / cm 2 . The cells were seeded and cultured in R medium, MC medium and SP medium for 48 hours (37 ° C., 5% CO 2 ), and the cell adhesion state was observed under a microscope. Micrographs are shown in FIG. 8 (R medium), FIG. 9 (MC medium), and FIG. 10 (SP medium).
ヒト脂肪細胞由来幹細胞（ＡＤＳＣ）（ＬＯＮＺＡ社、以下「ＡＤＳＣ」と言う）を、接着細胞用培養フラスコ（Ｎｕｎｃ ＥａｓＹＦｌａｓｋ Ｃｅｌｌ Ｃｕｌｔｕｒｅ Ｆｌａｓｋｓ、Ｔ２５、ｃａｔ：１５６３６７、コーティング剤未使用）に２０００ｃｅｌｌｓ／ｃｍ２で播種し、それぞれＲ培地、ＭＣ培地及びＳＰ培地で４８時間培養（３７℃、５％ＣＯ２）して細胞の接着状態を顕微鏡で観察した。顕微鏡写真を図８（Ｒ培地）、図９（ＭＣ培地）、図１０（ＳＰ培地）に示す。 [Example 10: Adhesion of adipose-derived mesenchymal stem cells]
Human fat cell-derived stem cells (ADSC) (LONZA, hereinafter referred to as "ADSC") were placed in a culture flask for adherent cells (Nunc EasYFlash Cell Culture Flashs, T25, cat: 156637, no coating agent used) at 2000 cells / cm 2 . The cells were seeded and cultured in R medium, MC medium and SP medium for 48 hours (37 ° C., 5% CO 2 ), and the cell adhesion state was observed under a microscope. Micrographs are shown in FIG. 8 (R medium), FIG. 9 (MC medium), and FIG. 10 (SP medium).
Ｒ培地では脂肪細胞由来幹細胞がフラスコに接着して、伸長しているのが観察できたが、他の無血清培地では、伸長している脂肪細胞由来幹細胞は観察できなかった。以上より、Ｒ培地は他の無血清培地に比べて細胞接着性を上げることが確認できた。
In the R medium, adipocyte-derived stem cells were observed to adhere to the flask and elongated, but in other serum-free media, the elongated adipocyte-derived stem cells could not be observed. From the above, it was confirmed that the R medium has higher cell adhesion than other serum-free media.
［実施例１１：ＴＨＰ－１マクロファージに対する影響］
ヒト急性単球性白血病由来細胞株ＴＨＰ－１（理研バイオリソースセンター、以下「ＴＨＰ－１」と言う）を推奨プロトコルに従い拡大培養し凍結ストックを作製した。ＴＨＰ－１の起眠播種より９日目に、ＴＨＰ－１懸濁液を回収して、ＴＨＰ－１懸濁液にＰｈｏｒｂｏｌ １２－Ｍｙｒｉｓｔａｔｅ １３－Ａｃｅｔａｔｅ（富士フィルム和光純薬社）を最終濃度１００ ｎＭとなるよう添加した。１２ウェルプレート（Ｃｏｒｎｉｎｇ Ｉｎｃ．社）の各ウェルに、ＴＨＰ－１懸濁液を１ｍＬずつ播種して培養（３７℃、５％ＣＯ２）を行いＴＨＰ－１の分化誘導を行った。ｈＵＣＭＳＣをそれぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＭＰ培地及びＳＰ培地で培養した後に、凍結保存した細胞を融解して生細胞濃度が８．９６×１０４ｃｅｌｌｓ／ｍＬとなるように、１０％ＦＢＳ－ＲＰＭＩ培地を加え、更に最終濃度１μｇ／ｍＬとなるようにＬＰＳ－ＥＢ（ＩｎｖｉｖｏＧｅｎ社）を加えた。なお、１０％ＦＢＳ－ＲＰＭＩ培地は、５０ｍＬのＦＢＳ及び５ｍＬのＰｅｎｉｃｉｌｌｉｎ－Ｓｔｒｅｐｔｏｍｙｃｉｎ， Ｌｉｑｕｉｄ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ Ｉｎｃ．社）を４４５ｍＬのＲＰＭＩ－１６４０ Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ Ｉｎｃ．社）に加えて調製した。ＴＨＰ－１分化誘導後３日目に、調製を行った細胞をウェルのトランズウェルインサート内に加えて２日間共培養を行った。共培養を開始してから２日後に、ＴＨＰ－１のｔｏｔａｌ ＲＮＡを回収し、ＣＣＬ２、ＴＮＦ及びＰＤＧＦＢのｍＲＮＡ発現量を定量ＰＣＲで測定した。各サンプルで得られたｍＲＮＡ発現量から式（１）を用いて、抑制率を求めた。なお、プライマーは表５に記載したものを用いた。結果を図１１（ＣＣＬ２）、図１２（ＴＮＦ）及び図１３（ＰＤＧＦＢ）に示した。
１００×（１－（各サンプルのｍＲＮＡ量）／（ＬＰＳ（＋）のｍＲＮＡ量））
・・・式（１）
[Example 11: Effect on THP-1 macrophages]
A human acute monocytic leukemia-derived cell line THP-1 (RIKEN BioResource Center, hereinafter referred to as "THP-1") was expanded and cultured according to the recommended protocol to prepare a frozen stock. On the 9th day from the dormant sowing of THP-1, the THP-1 suspension was collected, and the THP-1 suspension was liquidated with Phrol 12-Myristate 13-Actate (Fuji Film Wako Pure Chemical Industries, Ltd.) at a final concentration of 100. It was added so as to be nM. In each well of a 12-well plate (Corning Inc.), 1 mL of the THP-1 suspension was inoculated and cultured (37 ° C., 5% CO 2 ) to induce the differentiation of THP-1. After culturing hUCMSC in DMEM / F12 medium, R medium, SF medium, MP medium and SP medium, respectively, the cryopreserved cells are thawed so that the viable cell concentration becomes 8.96 × 10 4 cells / mL. 10% FBS-RPMI medium was added, and LPS-EB (InvivoGen) was further 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). On the 3rd day after the induction of THP-1 differentiation, the prepared cells were added into the Transwell insert of the well and co-cultured for 2 days. Two days after the start of co-culture, total RNA of THP-1 was collected, and the mRNA expression levels of CCL2, TNF and PDGFB were measured by quantitative PCR. From the mRNA expression level obtained in each sample, the suppression rate was determined using the formula (1). The primers shown in Table 5 were used. The results are shown in FIGS. 11 (CCL2), 12 (TNF) and 13 (PDGFB).
100 × (1- (amount of mRNA of each sample) / (amount of mRNA of LPS (+)))
... Equation (1)
ヒト急性単球性白血病由来細胞株ＴＨＰ－１（理研バイオリソースセンター、以下「ＴＨＰ－１」と言う）を推奨プロトコルに従い拡大培養し凍結ストックを作製した。ＴＨＰ－１の起眠播種より９日目に、ＴＨＰ－１懸濁液を回収して、ＴＨＰ－１懸濁液にＰｈｏｒｂｏｌ １２－Ｍｙｒｉｓｔａｔｅ １３－Ａｃｅｔａｔｅ（富士フィルム和光純薬社）を最終濃度１００ ｎＭとなるよう添加した。１２ウェルプレート（Ｃｏｒｎｉｎｇ Ｉｎｃ．社）の各ウェルに、ＴＨＰ－１懸濁液を１ｍＬずつ播種して培養（３７℃、５％ＣＯ２）を行いＴＨＰ－１の分化誘導を行った。ｈＵＣＭＳＣをそれぞれＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＭＰ培地及びＳＰ培地で培養した後に、凍結保存した細胞を融解して生細胞濃度が８．９６×１０４ｃｅｌｌｓ／ｍＬとなるように、１０％ＦＢＳ－ＲＰＭＩ培地を加え、更に最終濃度１μｇ／ｍＬとなるようにＬＰＳ－ＥＢ（ＩｎｖｉｖｏＧｅｎ社）を加えた。なお、１０％ＦＢＳ－ＲＰＭＩ培地は、５０ｍＬのＦＢＳ及び５ｍＬのＰｅｎｉｃｉｌｌｉｎ－Ｓｔｒｅｐｔｏｍｙｃｉｎ， Ｌｉｑｕｉｄ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ Ｉｎｃ．社）を４４５ｍＬのＲＰＭＩ－１６４０ Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ Ｉｎｃ．社）に加えて調製した。ＴＨＰ－１分化誘導後３日目に、調製を行った細胞をウェルのトランズウェルインサート内に加えて２日間共培養を行った。共培養を開始してから２日後に、ＴＨＰ－１のｔｏｔａｌ ＲＮＡを回収し、ＣＣＬ２、ＴＮＦ及びＰＤＧＦＢのｍＲＮＡ発現量を定量ＰＣＲで測定した。各サンプルで得られたｍＲＮＡ発現量から式（１）を用いて、抑制率を求めた。なお、プライマーは表５に記載したものを用いた。結果を図１１（ＣＣＬ２）、図１２（ＴＮＦ）及び図１３（ＰＤＧＦＢ）に示した。
１００×（１－（各サンプルのｍＲＮＡ量）／（ＬＰＳ（＋）のｍＲＮＡ量））
・・・式（１）
[Example 11: Effect on THP-1 macrophages]
A human acute monocytic leukemia-derived cell line THP-1 (RIKEN BioResource Center, hereinafter referred to as "THP-1") was expanded and cultured according to the recommended protocol to prepare a frozen stock. On the 9th day from the dormant sowing of THP-1, the THP-1 suspension was collected, and the THP-1 suspension was liquidated with Phrol 12-Myristate 13-Actate (Fuji Film Wako Pure Chemical Industries, Ltd.) at a final concentration of 100. It was added so as to be nM. In each well of a 12-well plate (Corning Inc.), 1 mL of the THP-1 suspension was inoculated and cultured (37 ° C., 5% CO 2 ) to induce the differentiation of THP-1. After culturing hUCMSC in DMEM / F12 medium, R medium, SF medium, MP medium and SP medium, respectively, the cryopreserved cells are thawed so that the viable cell concentration becomes 8.96 × 10 4 cells / mL. 10% FBS-RPMI medium was added, and LPS-EB (InvivoGen) was further 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). On the 3rd day after the induction of THP-1 differentiation, the prepared cells were added into the Transwell insert of the well and co-cultured for 2 days. Two days after the start of co-culture, total RNA of THP-1 was collected, and the mRNA expression levels of CCL2, TNF and PDGFB were measured by quantitative PCR. From the mRNA expression level obtained in each sample, the suppression rate was determined using the formula (1). The primers shown in Table 5 were used. The results are shown in FIGS. 11 (CCL2), 12 (TNF) and 13 (PDGFB).
100 × (1- (amount of mRNA of each sample) / (amount of mRNA of LPS (+)))
... Equation (1)
臍帯由来間葉系幹細胞と共培養することによって、ＴＨＰ－１マクロファージのＣＣＬ２、ＴＮＦ及びＰＤＧＦＢのｍＲＮＡ発現量が抑制された。なかでも、この効果はＲ培地で培養を行った臍帯由来間葉系幹細胞が最も高かった。
By co-culturing with umbilical cord-derived mesenchymal stem cells, the mRNA expression levels of CCL2, TNF and PDGFB of THP-1 macrophages were suppressed. Among them, this effect was highest in umbilical cord-derived mesenchymal stem cells cultured in R medium.
［培地特性］
ＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＳＰ培地及びＭＳ培地について、低粘度計（ローターＮｏ．０１、ＴＶＥ－２０Ｌ、東機産業社）を用いて、２０℃、１００ｒｐｍ、１８０秒における粘度を測定した。また、浸透圧及びＰＨも測定した（表６）。なお、浸透圧比は、第十七改正日本薬局方に基づき２８６ｍＯｓｍ（０．９ｗ／ｖ％塩化ナトリウム水溶液）の浸透圧に対する試料の浸透圧の比とし、浸透圧は日本薬局方記載の浸透圧測定法（氷点降下法）を参考にして測定した。なお、浸透圧比測定用標準液（０．９ｗ／ｖ％塩化ナトリウム水溶液）は、日本薬局方 生理食塩液 大塚生食注（大塚製薬工場製）を用いた。 [Medium characteristics]
For DMEM / F12 medium, R medium, SF medium, SP medium and MS medium, use a low viscometer (Rotor No. 01, TVE-20L, Toki Sangyo Co., Ltd.) to determine the viscosity at 20 ° C., 100 rpm, 180 seconds. It was measured. Osmolarity and pH were also measured (Table 6). The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 17th revised Japanese Pharmacopoeia, and the osmotic pressure is the osmotic pressure measurement described in the Japanese Pharmacopoeia. The measurement was performed with reference to the method (freezing point descent method). As the standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution), the Japanese Pharmacopoeia physiological saline solution Otsuka Raw Food Injection (manufactured by Otsuka Pharmaceutical Factory) was used.
ＤＭＥＭ／Ｆ１２培地、Ｒ培地、ＳＦ培地、ＳＰ培地及びＭＳ培地について、低粘度計（ローターＮｏ．０１、ＴＶＥ－２０Ｌ、東機産業社）を用いて、２０℃、１００ｒｐｍ、１８０秒における粘度を測定した。また、浸透圧及びＰＨも測定した（表６）。なお、浸透圧比は、第十七改正日本薬局方に基づき２８６ｍＯｓｍ（０．９ｗ／ｖ％塩化ナトリウム水溶液）の浸透圧に対する試料の浸透圧の比とし、浸透圧は日本薬局方記載の浸透圧測定法（氷点降下法）を参考にして測定した。なお、浸透圧比測定用標準液（０．９ｗ／ｖ％塩化ナトリウム水溶液）は、日本薬局方 生理食塩液 大塚生食注（大塚製薬工場製）を用いた。 [Medium characteristics]
For DMEM / F12 medium, R medium, SF medium, SP medium and MS medium, use a low viscometer (Rotor No. 01, TVE-20L, Toki Sangyo Co., Ltd.) to determine the viscosity at 20 ° C., 100 rpm, 180 seconds. It was measured. Osmolarity and pH were also measured (Table 6). The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 17th revised Japanese Pharmacopoeia, and the osmotic pressure is the osmotic pressure measurement described in the Japanese Pharmacopoeia. The measurement was performed with reference to the method (freezing point descent method). As the standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution), the Japanese Pharmacopoeia physiological saline solution Otsuka Raw Food Injection (manufactured by Otsuka Pharmaceutical Factory) was used.
いずれの無血清培地も、ＤＭＥＭ／Ｆ１２培地と比較して、粘度が低かったが、その中でもＲ培地が最も低かった。また、ＳＰ培地は、ＤＭＥＭ／Ｆ１２培地と同程度の浸透圧であったが、その他の無血清培地は、ＤＭＥＭ／Ｆ１２培地と比較して、浸透圧が低く、その中でもＲ培地が最も低かった。
以上の結果より、Ｒ培地が、低粘度、低浸透圧でありながら、細胞に強い接着性を持たせる可能性が示唆された。 All serum-free media had lower viscosities than DMEM / F12 media, but R medium had the lowest viscosity. The SP medium had the same osmotic pressure as the DMEM / F12 medium, but the other serum-free media had a lower osmotic pressure than the DMEM / F12 medium, and the R medium had the lowest osmotic pressure. ..
From the above results, it was suggested that the R medium may have strong adhesiveness to cells while having low viscosity and low osmotic pressure.
以上の結果より、Ｒ培地が、低粘度、低浸透圧でありながら、細胞に強い接着性を持たせる可能性が示唆された。 All serum-free media had lower viscosities than DMEM / F12 media, but R medium had the lowest viscosity. The SP medium had the same osmotic pressure as the DMEM / F12 medium, but the other serum-free media had a lower osmotic pressure than the DMEM / F12 medium, and the R medium had the lowest osmotic pressure. ..
From the above results, it was suggested that the R medium may have strong adhesiveness to cells while having low viscosity and low osmotic pressure.
［実施例１２：Ｔ細胞に対する影響１］
Ｒ培地もしくはＳＦ培地で培養した後に凍結保存されたｈＵＣＭＳＣを融解して２４ウェルプレートに、１．３３ｘ１０５ｃｅｌｌｓ／ウェルで播種し、１５時間以上の培養を行った。その後、カルボキシフルオレセインスクシンイミジルエステル（５－（ａｎｄ －６）－Ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ ｄｉａｃｅｔａｔｅ ｓｕｃｃｉｎｉｍｉｄｙｌ ｅｓｔｅｒ、以下「ＣＦＳＥ」と言う）染色を行い抗ＣＤ３／ＣＤ２８抗体で増殖刺激した４．００ｘ１０５ｃｅｌｌｓ／ｗｅｌｌの末梢血単核細胞（Ｐｅｒｉｐｈｅｒａｌ Ｂｌｏｏｄ Ｍｏｎｏｎｕｃｌｅａｒ Ｃｅｌｌｓ、以下「ＰＢＭＣ」と言う）と４日間共培養した。共培養の後、ＰＢＭＣを回収し、フローサイトメーターを用いてＣＦＳＥ染色の蛍光強度により増殖したＴ細胞の割合を計測した。なお、解析の対象はＣＤ４陽性およびＣＤ８陽性Ｔ細胞とし、これらＴ細胞はＣＤ３、ＣＤ４及びＣＤ８の抗体染色により同定した。また、増殖したＴ細胞の割合は、抗ＣＤ３／ＣＤ２８抗体刺激無し群の増殖率が０．５％前後となる位置にゲートを設定し、同様のゲートを抗ＣＤ３／ＣＤ２８抗体刺激した試験群に適応させることで測定した。なお、図には示していないが、陽性対照としてヒト脂肪由来間葉系細胞（ｈＡＤＭＳＣ）を用いた。抗ＣＤ３／ＣＤ２８抗体で刺激しＭＳＣとの共培養を行っていない群（ＣＮＴＬ）に比べて、ｈＵＣＭＳＣと共培養した群では、増殖したＣＤ４陽性Ｔ細胞およびＣＤ８陽性Ｔ細胞の割合は減少した（図１４及び図１５）。また、Ｒ培地で培養したｈＵＣＭＳＣに比べ、ＳＦ培地で培養したｈＵＣＭＳＣの増殖抑制効果は低かった。 [Example 12: Effect on T cells 1]
In 24-well plates were thawed cryopreserved hUCMSC after culturing in R media or SF medium, and plated at 1.33 × 10 5 cells / well, were cultured for 15 hours or more. Then, carboxyfluorescein succinimidyl ester (5- (and-6) -Carboxyfluorescein diacetate succinidiyl ester, hereinafter referred to as "CFSE") was stained and proliferated with an anti-CD3 / CD28 antibody . Peripheral of 4.00x10 5 cells / well. It was co-cultured with blood mononuclear cells (Peripheral Blood Mononuclear Cells, hereinafter referred to as "PBMC") for 4 days. After co-culture, PBMCs were collected and the proportion of T cells proliferated by the fluorescence intensity of CFSE staining was measured using a flow cytometer. The subjects of analysis were CD4 positive and CD8 positive T cells, and these T cells were identified by antibody staining of CD3, CD4 and CD8. Regarding the proportion of proliferated T cells, a gate was set at a position where the proliferation rate of the anti-CD3 / CD28 antibody-stimulated group was around 0.5%, and the same gate was used for the test group stimulated with anti-CD3 / CD28 antibody. Measured by adaptation. Although not shown in the figure, human adipose-derived mesenchymal cells (hADMSC) were used as positive controls. The proportion of proliferated CD4-positive T cells and CD8-positive T cells decreased in the group co-cultured with hUCMSC compared to the group not co-cultured with MSC stimulated with anti-CD3 / CD28 antibody (CNTL). 14 and 15). In addition, the growth inhibitory effect of hUCMSC cultured in SF medium was lower than that of hUCMSC cultured in R medium.
Ｒ培地もしくはＳＦ培地で培養した後に凍結保存されたｈＵＣＭＳＣを融解して２４ウェルプレートに、１．３３ｘ１０５ｃｅｌｌｓ／ウェルで播種し、１５時間以上の培養を行った。その後、カルボキシフルオレセインスクシンイミジルエステル（５－（ａｎｄ －６）－Ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ ｄｉａｃｅｔａｔｅ ｓｕｃｃｉｎｉｍｉｄｙｌ ｅｓｔｅｒ、以下「ＣＦＳＥ」と言う）染色を行い抗ＣＤ３／ＣＤ２８抗体で増殖刺激した４．００ｘ１０５ｃｅｌｌｓ／ｗｅｌｌの末梢血単核細胞（Ｐｅｒｉｐｈｅｒａｌ Ｂｌｏｏｄ Ｍｏｎｏｎｕｃｌｅａｒ Ｃｅｌｌｓ、以下「ＰＢＭＣ」と言う）と４日間共培養した。共培養の後、ＰＢＭＣを回収し、フローサイトメーターを用いてＣＦＳＥ染色の蛍光強度により増殖したＴ細胞の割合を計測した。なお、解析の対象はＣＤ４陽性およびＣＤ８陽性Ｔ細胞とし、これらＴ細胞はＣＤ３、ＣＤ４及びＣＤ８の抗体染色により同定した。また、増殖したＴ細胞の割合は、抗ＣＤ３／ＣＤ２８抗体刺激無し群の増殖率が０．５％前後となる位置にゲートを設定し、同様のゲートを抗ＣＤ３／ＣＤ２８抗体刺激した試験群に適応させることで測定した。なお、図には示していないが、陽性対照としてヒト脂肪由来間葉系細胞（ｈＡＤＭＳＣ）を用いた。抗ＣＤ３／ＣＤ２８抗体で刺激しＭＳＣとの共培養を行っていない群（ＣＮＴＬ）に比べて、ｈＵＣＭＳＣと共培養した群では、増殖したＣＤ４陽性Ｔ細胞およびＣＤ８陽性Ｔ細胞の割合は減少した（図１４及び図１５）。また、Ｒ培地で培養したｈＵＣＭＳＣに比べ、ＳＦ培地で培養したｈＵＣＭＳＣの増殖抑制効果は低かった。 [Example 12: Effect on T cells 1]
In 24-well plates were thawed cryopreserved hUCMSC after culturing in R media or SF medium, and plated at 1.33 × 10 5 cells / well, were cultured for 15 hours or more. Then, carboxyfluorescein succinimidyl ester (5- (and-6) -Carboxyfluorescein diacetate succinidiyl ester, hereinafter referred to as "CFSE") was stained and proliferated with an anti-CD3 / CD28 antibody . Peripheral of 4.00x10 5 cells / well. It was co-cultured with blood mononuclear cells (Peripheral Blood Mononuclear Cells, hereinafter referred to as "PBMC") for 4 days. After co-culture, PBMCs were collected and the proportion of T cells proliferated by the fluorescence intensity of CFSE staining was measured using a flow cytometer. The subjects of analysis were CD4 positive and CD8 positive T cells, and these T cells were identified by antibody staining of CD3, CD4 and CD8. Regarding the proportion of proliferated T cells, a gate was set at a position where the proliferation rate of the anti-CD3 / CD28 antibody-stimulated group was around 0.5%, and the same gate was used for the test group stimulated with anti-CD3 / CD28 antibody. Measured by adaptation. Although not shown in the figure, human adipose-derived mesenchymal cells (hADMSC) were used as positive controls. The proportion of proliferated CD4-positive T cells and CD8-positive T cells decreased in the group co-cultured with hUCMSC compared to the group not co-cultured with MSC stimulated with anti-CD3 / CD28 antibody (CNTL). 14 and 15). In addition, the growth inhibitory effect of hUCMSC cultured in SF medium was lower than that of hUCMSC cultured in R medium.
［実施例１３：Ｔ細胞に対する影響２］
ＤＭＥＭ／Ｆ１２培地で培養した後に凍結保存されたｈＵＣＭＳＣを融解して実施例１２と同様に増殖したＴ細胞の割合を計測した。抗ＣＤ３／ＣＤ２８抗体で刺激しｈＵＣＭＳＣとの共培養を行っていない群（ＣＮＴＬ）に比べて、ｈＵＣＭＳＣと共培養した群では、増殖したＣＤ４陽性Ｔ細胞およびＣＤ８陽性Ｔ細胞の割合は減少した（図１６及び図１７）。実施例１２及び実施例１３の陽性対照との比較から、Ｒ培地で培養したｈＵＣＭＳＣのＴ細胞増殖抑制効果は、ＤＭＥＭ／Ｆ１２培地で培養したｈＵＣＭＳＣのＴ細胞増殖抑制効果に比べて高いことがわかった。 [Example 13: Effect on T cells 2]
After culturing in DMEM / F12 medium, hUCMSC cryopreserved was thawed and the proportion of T cells proliferated in the same manner as in Example 12 was measured. The proportion of proliferated CD4-positive T cells and CD8-positive T cells decreased in the group co-cultured with hUCMSC compared to the group not co-cultured with hUCMSC stimulated with anti-CD3 / CD28 antibody (CNTL). 16 and 17). From the comparison with the positive controls of Examples 12 and 13, it was found that the T cell growth inhibitory effect of hUCMSC cultured in R medium was higher than the T cell proliferation inhibitory effect of hUCMSC cultured in DMEM / F12 medium. rice field.
ＤＭＥＭ／Ｆ１２培地で培養した後に凍結保存されたｈＵＣＭＳＣを融解して実施例１２と同様に増殖したＴ細胞の割合を計測した。抗ＣＤ３／ＣＤ２８抗体で刺激しｈＵＣＭＳＣとの共培養を行っていない群（ＣＮＴＬ）に比べて、ｈＵＣＭＳＣと共培養した群では、増殖したＣＤ４陽性Ｔ細胞およびＣＤ８陽性Ｔ細胞の割合は減少した（図１６及び図１７）。実施例１２及び実施例１３の陽性対照との比較から、Ｒ培地で培養したｈＵＣＭＳＣのＴ細胞増殖抑制効果は、ＤＭＥＭ／Ｆ１２培地で培養したｈＵＣＭＳＣのＴ細胞増殖抑制効果に比べて高いことがわかった。 [Example 13: Effect on T cells 2]
After culturing in DMEM / F12 medium, hUCMSC cryopreserved was thawed and the proportion of T cells proliferated in the same manner as in Example 12 was measured. The proportion of proliferated CD4-positive T cells and CD8-positive T cells decreased in the group co-cultured with hUCMSC compared to the group not co-cultured with hUCMSC stimulated with anti-CD3 / CD28 antibody (CNTL). 16 and 17). From the comparison with the positive controls of Examples 12 and 13, it was found that the T cell growth inhibitory effect of hUCMSC cultured in R medium was higher than the T cell proliferation inhibitory effect of hUCMSC cultured in DMEM / F12 medium. rice field.
本発明により、細胞の接着性が高く、細胞の増殖速度が速い間葉系幹細胞を提供できる。また、本発明の間葉系幹細胞は、マクロファージのＣＣＬ２、ＴＮＦ及びＰＤＧＦＢのｍＲＮＡ発現を低下させ、また活性化したＴ細胞の増殖を抑制する作用も有し、顕著な抗炎症効果を奏する。
INDUSTRIAL APPLICABILITY According to the present invention, mesenchymal stem cells having high cell adhesion and a high cell proliferation rate can be provided. In addition, the mesenchymal stem cells of the present invention have an effect of reducing the mRNA expression of macrophages CCL2, TNF and PDGFB and suppressing the proliferation of activated T cells, and exert a remarkable anti-inflammatory effect.
Claims (4)
- 顆粒球コロニー形成刺激因子（Ｇ－ＣＳＦ（Ｇｒａｎｕｌｏｃｙｔｅ Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ））の産生（Ｐｒｏｄｕｃｔｉｏｎ）量が亢進していることを特徴とする、間葉系幹細胞。 A mesenchymal stem cell characterized in that the amount of production (Production) of a granulocyte colony-stimulating factor (G-CSF (Granulocyte Colony Stimulating Factor)) is increased.
- エオタキシン（ＥＯＴＡＸＩＮ）、フラクタルカイン（ＦＲＡＣＴＡＬＫＩＮＥ）、ＧＲＯ、ＭＣＰ－３及びＶＥＧＦからなる群より選択される少なくとも一つの産生量が亢進していることを特徴とする、間葉系幹細胞。 A mesenchymal stem cell characterized in that the production amount of at least one selected from the group consisting of EOTAXIN, FRACTALKINE, GRO, MCP-3 and VEGF is enhanced.
- 脂肪組織由来、臍帯組織由来、もしくは骨髄組織由来である、請求項１又は２に記載の間葉系幹細胞。 The mesenchymal stem cell according to claim 1 or 2, which is derived from adipose tissue, umbilical cord tissue, or myeloid tissue.
- 請求項１から３のいずれか１項に記載の間葉系幹細胞を誘導する細胞用培地。 A cell medium for inducing mesenchymal stem cells according to any one of claims 1 to 3.
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