WO2018030448A1 - Cell culture medium and method for culturing cells - Google Patents
Cell culture medium and method for culturing cells Download PDFInfo
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- WO2018030448A1 WO2018030448A1 PCT/JP2017/028861 JP2017028861W WO2018030448A1 WO 2018030448 A1 WO2018030448 A1 WO 2018030448A1 JP 2017028861 W JP2017028861 W JP 2017028861W WO 2018030448 A1 WO2018030448 A1 WO 2018030448A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/24—Mucus; Mucous glands; Bursa; Synovial fluid; Arthral fluid; Excreta; Spinal fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- the present invention relates to a cell culture medium and a cell culture method, and more particularly to a cell culture medium used for culturing mesenchymal stem cells and a cell culture method of mesenchymal stem cells.
- stem cells have self-replicating ability and pluripotency, and are assumed as materials for cells and tissues in regenerative medicine.
- stem cells are roughly divided into two types, pluripotent stem cells and tissue stem cells, and are present in various tissues of the living body.
- mesenchymal stem cells among tissue stem cells are known to be contained in bone marrow fluid, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, etc.
- hematopoietic stem cells are mainly contained in bone marrow fluid, It is also known to exist in peripheral blood and umbilical cord blood.
- the present inventors have previously clarified that mesenchymal stem cells isolated from bone marrow fluid are effective in treating patients with liver diseases. Specifically, after collecting bone marrow fluid from a patient with liver disease, separating and concentrating components containing mesenchymal stem cells from it to obtain a bone marrow cell-containing preparation for liver regeneration, the preparation is given to the patient again. Intravenous administration improved liver disease and restored liver function (Patent Document 1).
- stem cells contained in various tissues are extremely small.
- mesenchymal stem cells have a prevalence of about 0.05% in bone marrow fluid, and a large amount of bone marrow fluid is collected for direct clinical use. This is necessary and the burden on the living body is increased. For this reason, it is desirable to use subcultured stem cells collected from living tissue.
- the present inventors tried to proliferate mesenchymal stem cells to a target amount by collecting bone marrow fluid from a patient with liver disease and culturing it.
- the present inventors originally separated mesenchymal stem cells from the collected bone marrow fluid and then cultured them using a commercially available medium.
- the whole bone marrow cells in the bone marrow fluid were not separated without separating the mesenchymal stem cells.
- the present inventors have found that, among various proteins contained in the culture medium in which whole bone marrow cells are cultured, a specific cytokine is effective for cell proliferation. That is, it has been found that by using a medium containing a specific cytokine, it is possible to grow a large number of cells in a shorter time even with a small amount of cells, and the present invention has been completed.
- the present invention is as follows. 1. A medium for cell culture, which contains MIG and I-309. 2. 2. The cell culture medium according to 1 above, wherein the cells are mesenchymal stem cells. 3. 3. The cell culture medium according to 2 above, wherein the mesenchymal stem cells are derived from bone marrow fluid. 4). 4. The cell culture medium according to any one of 1 to 3, further comprising IL-8 and / or MIP-1 ⁇ . 5). A cell culture method in which cells are grown and cultured in a medium containing MIG and I-309. 6). 6. The cell culture method according to 5 above, wherein the cell is a mesenchymal stem cell. 7). 7. The cell culture method according to 6 above, wherein the mesenchymal stem cells are derived from bone marrow fluid. 8). 8. The cell culture method according to any one of 5 to 7, wherein the medium further contains IL-8 and / or MIP-1 ⁇ .
- the medium of the present invention containing a specific cytokine By using the medium of the present invention containing a specific cytokine, a large number of cells can be proliferated in a shorter time even with a small amount of cells.
- the amount of cells collected from the patient can be reduced. Therefore, treatment can be performed while reducing the burden on the patient.
- FIG. 1 is a graph showing the results of MTS assay for human whole bone marrow cells cultured in Test Example 1.
- FIG. 2 is a graph showing a growth curve of human bone marrow mesenchymal stem cells cultured in Examples.
- FIG. 3 is a graph showing the results of MTS assay for human bone marrow mesenchymal stem cells and the like cultured in Examples.
- FIG. 4 is a graph showing a growth curve of human bone marrow mesenchymal stem cells cultured in Reference Example.
- FIG. 5 is a photographic diagram showing that human bone marrow mesenchymal stem cells cultured in Examples differentiated into adipocytes.
- the present invention provides a medium for cell culture, which is a medium for cell culture and contains MIG and I-309.
- the basal medium that can be used for the cell culture medium of the present invention is not particularly limited, but includes amino acids, vitamins, inorganic salts, and the like, which are essential components for normal cell growth.
- Examples of the basal medium include Eagle basal medium (MEM), Alpha Eagle basal medium ( ⁇ MEM), Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium.
- basic medium in the present specification refers to a medium before MIG, I-309 and the like are added to the cell culture medium of the present invention, and corresponds to the above-described commercially available basic medium and the like.
- the cell culture medium of the present invention contains at least MIG and I-309.
- MIG is a protein, a kind of cytokine, and one of the CXC chemokine family also called CXCL9. MIG is known to be produced from monocytes, macrophages, and endothelial cells stimulated with interferon ⁇ and exhibit chemotaxis of Th1 lymphocytes. It is also said to inhibit tumor cell growth, angiogenesis, and hematopoietic stem progenitor cell colony formation. In the present invention, for example, MIG manufactured by Wako Pure Chemical Industries, Ltd. can be used.
- I-309 is a protein, a kind of cytokine, and is one of the CC chemokine family also called CCL1. Produced by T lymphocytes, monocytes and mast cells, it is said to induce neutrophils, macrophages and vascular smooth muscle cells and promote the proliferation of some cells.
- I-309 manufactured by Wako Pure Chemical Industries, Ltd. can be used.
- the cell culture medium of the present invention preferably contains 100 to 1000 ng / mL, more preferably 375 to 500 ng / mL of MIG with respect to the entire medium. If it exceeds 1000 ng / mL, the culture cost may increase.
- the cell culture medium of the present invention preferably contains 50 to 1000 ng / mL, more preferably 100 to 500 ng / mL of I-309 with respect to the whole medium. If it exceeds 1000 ng / mL, the culture cost may increase.
- the cell culture medium of the present invention preferably contains MIG and I-309 at a mass ratio of 1: 0.05 to 1: 1, and 1: 0.05 to 1: 0.15. It is more preferable to contain. By being in the above range, a higher cell proliferation promoting effect can be obtained.
- the cell culture medium of the present invention may further contain IL-8 and / or MIP-1 ⁇ as another cytokine.
- IL-8 and MIP-1 ⁇ are also classified as chemokines, which are a kind of cytokine, and IL-8 is classified as a CXC chemokine and MIP-1 ⁇ is classified as a CC chemokine.
- IL-8 and MIP-1 ⁇ manufactured by Wako Pure Chemical Industries, Ltd. can be used.
- the cell culture medium of the present invention may also contain other components, in addition to the above, depending on the type and purpose of the cells to be cultured as long as the effects of the present invention are not impaired.
- cytokines examples include, for example, MCP3, RANTES, MIP-1 ⁇ , IL-6, IL-10, MCP2, CCL24, GRO, uPAR, M-CSF, NAP-2, sgp130, TIMP1, ENA-78, OPG, Examples include IGFBP2, ANG, IL-7, and VEGF-A.
- the cell culture medium of the present invention may contain nutrients, serum, antibiotics, and the like depending on the type and purpose of the cells to be cultured within a range that does not impair the effects of the present invention.
- Examples of nutritional components include fatty acids and vitamins.
- heterologous serum and allogeneic serum can be used as the serum.
- the heterologous serum means serum derived from a different species of organism from the recipient when the cell culture is used for cell medicine or the like.
- serum derived from bovine or horse for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum.
- FBS, FCS fetal calf serum
- CS calf serum
- HS horse serum
- Allogeneic serum means serum derived from the same species of organism as the recipient.
- human serum corresponds to allogeneic serum.
- Allogeneic serum includes autoserum (also called autologous serum), ie, serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient.
- antibiotics examples include penicillin, streptomycin, gentamicin and the like.
- the animal species of the cells cultured in the medium of the present invention is not particularly limited, and for example, humans, rats, mice, pigs and the like can be used according to the use of the cells.
- the type of cells to be cultured in the medium of the present invention is not particularly limited. For example, it is included in the process of differentiation from ectodermal cells, mesoderm cells, endoderm cells, and fertilized eggs into these cells. Cells, embryonic stem cells and somatic stem cells.
- ectoderm cells include neuronal cells, astrocyte cells, oligodendrocyte cells, and neural stem cells that are these stem cells.
- mesoderm cells include vascular cells, hematopoietic cells, mesenchymal cells, and stem cells thereof.
- hematopoietic cells examples include hematopoietic stem cells, hematopoietic progenitor cells, red blood cells, lymphocyte cells, granulocyte cells, and platelet cells.
- mesenchymal cells examples include bone cells, chondrocytes, muscle cells, cardiomyocytes, tendon cells, adipocytes, dermal papilla cells, dental pulp cells, and mesenchymal stem cells that are these stem cells.
- mesenchymal stem cells for example, those contained in bone marrow fluid, adipose tissue, placenta tissue, umbilical cord tissue, dental pulp, etc. can be used, and preferably mesenchymal stem cells derived from bone marrow fluid.
- endoderm cells include hepatocytes, pancreatic exocrine cells, pancreatic endocrine cells, gallbladder cells, and stem cells thereof.
- the cells are preferably stem cells, more preferably mesenchymal stem cells.
- the mesenchymal stem cell may be derived from any tissue such as bone marrow fluid, adipose tissue, or umbilical cord blood, for example, from the viewpoint of the effect of the present invention and from the viewpoint that more cells can be collected. Therefore, it is preferably a mesenchymal stem cell derived from bone marrow fluid.
- the present invention provides a cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
- the cell culture method of the present invention is performed using a medium containing MIG and I-309, that is, the above-described medium of the present invention.
- the cells to be cultured are described as mesenchymal stem cells, but the cell culture method of the present invention is not limited to mesenchymal stem cells.
- cell culture conditions and the like preferable conditions can be appropriately adopted depending on the type of cells.
- the method for obtaining mesenchymal stem cells is not particularly limited and can be appropriately selected according to the purpose.
- a method obtained by isolating from a solid such as human or mouse, each cell already cloned. Can be obtained from various institutions.
- a cell when a cell is cultured using the medium of the present invention and returned to the body of patients with various diseases, it is preferable to use cells collected from the body of the patient.
- mesenchymal stem cells when mesenchymal stem cells are administered to a patient to treat a patient with liver disease, a small amount of a target solution containing mesenchymal stem cells of the bone marrow of the patient is collected, and the medium of the present invention It is preferable to perform cell proliferation by culturing in
- erythrocytes are precipitated using PBS (phosphate buffered saline), HES (erythrocyte sedimentation agent) or the like in a tube into which bone marrow fluid has been dispensed. Thereafter, the supernatant is extracted and centrifuged to separate the target liquid containing mesenchymal stem cells.
- PBS phosphate buffered saline
- HES erythrocyte sedimentation agent
- mesenchymal stem cells When culturing mesenchymal stem cells, it is cultured in an incubator, for example, under conditions of a temperature of 37 ° C. and 5% CO 2 . As for the culture time, it is preferable to subculture before becoming confluent.
- the number of passages of mesenchymal stem cells is not particularly limited and can be appropriately selected depending on the purpose.
- the cell culture solution obtained by the cell culture method of the present invention or the cell fraction obtained by separating only cells from the cell culture solution can be used for various cell medicines, for example, depending on the cell type.
- mesenchymal stem cells as described above, it can be used for the treatment of patients with liver diseases.
- Test Example 1 Identification of cytokines (1) In Test Example 1, in order to identify the type of cytokine contained in the culture medium in which human whole bone marrow cells were cultured, human whole bone marrow cells were cultured as follows, and a cytokine array was obtained from the obtained culture medium supernatant. Analysis was carried out.
- ⁇ Culture of human whole bone marrow cells In order to culture human whole bone marrow cells including hematopoietic cells that are non-adherent cells and mesenchymal stem cells that are adherent cells, a culture dish that can simultaneously culture non-adherent cells and adherent cells (hereinafter, culture dish) A). As the culture dish A, a culture dish surface coated with nano-sized polymer and clay was used. By coating the culture dish in this way, cells that do not adhere in normal culture dishes can be adhered, for example, by increasing the cell adhesion of the culture surface. Therefore, culturing blood cells and mesenchymal stem cells simultaneously Can do.
- culture dish a culture dish surface coated with nano-sized polymer and clay was used.
- human whole bone marrow cells (Lonza BMSC, 2M-125D) were prepared. Culturing was performed under conditions of a temperature of 37 ° C. and 5% CO 2 .
- a culture dish IWAKI tissue culture dish 3000-035: culture dish B
- Dulbecco's modified Eagle medium DMEM
- fetal bovine serum fetal bovine serum
- gentamicin hereinafter referred to as medium B
- Test Example 2 Identification of cytokines (2) In Test Example 2, a test was performed to identify cytokines having a cell growth promoting effect among the 19 types of cytokines identified in Test Example 1.
- DMEM + Gentamycin + 10% FBS medium (basal medium) was prepared.
- This basal medium was prepared according to the formulation shown in Table 2 below.
- a “cytokine-added medium 1” was prepared by adding all 19 types of cytokines whose high concentrations were observed in Test Example 1 to the basal medium according to the formulation shown in Table 3 below.
- a “cytokine-added medium 2” was prepared by adding 4 of the 19 types of cytokines to the basal medium according to the formulation shown in Table 4 below, and used as the medium of Example 2.
- a “cytokine-added medium 3” was prepared by adding 2 of the 19 types of cytokines to the basal medium in accordance with the formulation shown in Table 5 below, and used as the medium of Example 3.
- the cells were cultured under conditions of 5% CO 2 and 37 ° C., and using the Incubate (registered trademark) ZOOM live cell imaging system (manufactured by Essen BioScience), the cells occupied in the bottom area of each well The ratio was calculated over time, and after culturing for 5 days, proliferation curves of human bone marrow mesenchymal stem cells were prepared. The results are shown in FIG.
- MTS assay was performed on the cytokine-added medium 1 (Example 1), the cytokine-added medium 3 (Example 3), and the control medium (basal medium) after culture. MTS assay was measured in the same manner as in Test Example 1. The result is shown in FIG.
- the cytokine-added medium 3 supplemented with MIG and I-309 showed the same effect of promoting proliferation of human bone marrow mesenchymal stem cells as the cytokine-added medium 1 supplemented with all 19 types of cytokines. Further, in the cytokine-added medium 2 to which IL-8 and MIP-1 ⁇ were added in addition to MIG and I-309, the same growth promoting effect as that obtained by adding all 19 types of cytokines was observed.
- Example 3 A medium was prepared in the same manner as in Example 1 except that the amount of MIG and I-309 added was 0 in the medium of Example 1 of Test Example 2, and this was used as the medium of Reference Example 1. That is, the medium of Reference Example 1 was prepared by adding 17 types of cytokines obtained by removing 2 types of cytokines, MIG and I-309, from all 19 types of cytokines to the basal medium. Then, human bone marrow mesenchymal stem cells were cultured in the same manner as in Test Example 2 using the medium of Reference Example 1, and a growth curve was prepared. Similarly to Test Example 2, a test was similarly performed on a medium to which all 19 types of cytokines were added (the medium in Example 1) and a medium to which no cytokine was added (control medium). The results are shown in FIG.
- the growth-promoting effect was significantly reduced in the medium in which 17 types of cytokines except MIG and I-309 were added from all 19 types of cytokines, compared to the medium in which all 19 types of cytokines were added. . From this, it was confirmed that MIG and I-309 have an effect of promoting cell proliferation.
- the ability of human bone marrow mesenchymal stem cells to differentiate into adipocytes was confirmed by the observation of adipocytes in the medium. That is, it can be confirmed that human bone marrow mesenchymal stem cells having differentiation potential can be proliferated and cultured by culturing human bone marrow mesenchymal stem cells using cytokine-added medium 3 supplemented with MIG and I-309. It was.
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Abstract
The object of the present invention is to provide a medium containing a specific cytokine and thus capable of growing and culturing many cells in a short period of time. The present invention pertains to a cell culture medium for proliferating and culturing cells, the medium containing MIG and I-309.
Description
本発明は、細胞培養用培地、及び細胞培養方法に関し、特に間葉系幹細胞の培養に用いる細胞培養用培地、及び間葉系幹細胞の細胞培養方法に関する。
The present invention relates to a cell culture medium and a cell culture method, and more particularly to a cell culture medium used for culturing mesenchymal stem cells and a cell culture method of mesenchymal stem cells.
細胞医療の分野において、細胞を利用して人体の各組織の治療を行うことが注目を浴びている。特に幹細胞は、自己複製能と多分化能を有しており、再生医療における細胞や組織の材料として想定されている。
In the field of cell medicine, it is attracting attention to treat each tissue of the human body using cells. In particular, stem cells have self-replicating ability and pluripotency, and are assumed as materials for cells and tissues in regenerative medicine.
これらの幹細胞は、多能性幹細胞と組織幹細胞の２つに大きく分けられ、生体の様々な組織に存在する。例えば、組織幹細胞のうち間葉系幹細胞は、骨髄液、脂肪組織、胎盤組織、臍帯組織、又は歯髄等に含まれていることが知られており、造血幹細胞は主に骨髄液に含まれ、末梢血、臍帯血にも存在することが知られている。
These stem cells are roughly divided into two types, pluripotent stem cells and tissue stem cells, and are present in various tissues of the living body. For example, mesenchymal stem cells among tissue stem cells are known to be contained in bone marrow fluid, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, etc., hematopoietic stem cells are mainly contained in bone marrow fluid, It is also known to exist in peripheral blood and umbilical cord blood.
細胞医療の分野では治療に用いる細胞を患者に投与する場合、生体から採取した後、細胞の培養を行わずに患者に投与する方法もあれば、一度生体外で培養した後に再び患者に投与する方法もある。
In the field of cell medicine, when cells to be used for treatment are administered to a patient, there is a method in which the cells are collected from a living body and then administered to the patient without culturing the cells. There is also a method.
前者の方法を用いて、本発明者らは、これまでに、骨髄液から分離した間葉系幹細胞が肝疾患の患者の治療に有効であることを明らかにしている。具体的には、肝疾患の患者から骨髄液を採取し、そこから間葉系幹細胞を含む成分を分離、濃縮して肝再生用骨髄細胞含有製剤を得た後に、当該製剤を再度その患者に点滴投与することにより、肝疾患を改善させ、肝機能を回復させることができた（特許文献１）。
Using the former method, the present inventors have previously clarified that mesenchymal stem cells isolated from bone marrow fluid are effective in treating patients with liver diseases. Specifically, after collecting bone marrow fluid from a patient with liver disease, separating and concentrating components containing mesenchymal stem cells from it to obtain a bone marrow cell-containing preparation for liver regeneration, the preparation is given to the patient again. Intravenous administration improved liver disease and restored liver function (Patent Document 1).
一方、各種組織に含まれる幹細胞の量は極めて少なく、例えば間葉系幹細胞は、骨髄液中における存在率が０.０５％程度であり、直接臨床に使用するには骨髄液を大量に採取する必要があり、生体への負担が大きくなってしまう。そのため、生体組織から採取した幹細胞は継代培養して用いることが望ましいとされている。
On the other hand, the amount of stem cells contained in various tissues is extremely small. For example, mesenchymal stem cells have a prevalence of about 0.05% in bone marrow fluid, and a large amount of bone marrow fluid is collected for direct clinical use. This is necessary and the burden on the living body is increased. For this reason, it is desirable to use subcultured stem cells collected from living tissue.
なお、間葉系幹細胞の培養について、採取した骨髄液の初代培養、継代培養を行い、目的とする量まで間葉系幹細胞を増殖させることは、従来既に公知である（特許文献２）。
In addition, regarding the mesenchymal stem cell culture, it is already known in the art to perform primary culture and subculture of the collected bone marrow fluid to proliferate the mesenchymal stem cells to a target amount (Patent Document 2).
そこで本発明者らは、肝疾患の患者から骨髄液を採取し、培養することによって、目的とする量にまで間葉系幹細胞を増殖することを試みた。本発明者らは当初、採取した骨髄液から間葉系幹細胞を分離した後に、市販の培地を用いて培養を行っていたが、間葉系幹細胞を分離せずに骨髄液中の全骨髄細胞をそのまま培養したところ、驚くべきことに、増殖させたい間葉系幹細胞のみを分離、培養した場合と比較して、より短時間に多くの細胞を培養できることを知見した。
Therefore, the present inventors tried to proliferate mesenchymal stem cells to a target amount by collecting bone marrow fluid from a patient with liver disease and culturing it. The present inventors originally separated mesenchymal stem cells from the collected bone marrow fluid and then cultured them using a commercially available medium. However, the whole bone marrow cells in the bone marrow fluid were not separated without separating the mesenchymal stem cells. As a result, it was surprisingly found that more cells can be cultured in a shorter time than when only mesenchymal stem cells to be proliferated are isolated and cultured.
本発明者らは、さらに鋭意研究を進めたところ、全骨髄細胞を培養した培養液に含まれる種々のタンパク質のうち、特定のサイトカインが、細胞の増殖に有効であることを見出した。すなわち、特定のサイトカインを含有する培地を用いることにより、少量の細胞であってもより短時間にかつ多くの細胞を増殖させることが可能となることを見出し、本発明を完成させた。
As a result of further diligent research, the present inventors have found that, among various proteins contained in the culture medium in which whole bone marrow cells are cultured, a specific cytokine is effective for cell proliferation. That is, it has been found that by using a medium containing a specific cytokine, it is possible to grow a large number of cells in a shorter time even with a small amount of cells, and the present invention has been completed.
すなわち、本発明は以下の通りである。
１．細胞を増殖培養するための培地であって、ＭＩＧ及びＩ－３０９を含有する、細胞培養用培地。
２．前記細胞が間葉系幹細胞である、前記１に記載の細胞培養用培地。
３．前記間葉系幹細胞が骨髄液由来である、前記２に記載の細胞培養用培地。
４．さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、前記１～３のいずれか１に記載の細胞培養用培地。
５．細胞を、ＭＩＧ及びＩ－３０９を含有する培地で増殖培養する、細胞培養方法。
６．前記細胞が間葉系幹細胞である、前記５に記載の細胞培養方法。
７．前記間葉系幹細胞が骨髄液由来である、前記６に記載の細胞培養方法。
８．前記培地が、さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、前記５～７のいずれか１に記載の細胞培養方法。 That is, the present invention is as follows.
1. A medium for cell culture, which contains MIG and I-309.
2. 2. The cell culture medium according to 1 above, wherein the cells are mesenchymal stem cells.
3. 3. The cell culture medium according to 2 above, wherein the mesenchymal stem cells are derived from bone marrow fluid.
4). 4. The cell culture medium according to any one of 1 to 3, further comprising IL-8 and / or MIP-1α.
5). A cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
6). 6. The cell culture method according to 5 above, wherein the cell is a mesenchymal stem cell.
7). 7. The cell culture method according to 6 above, wherein the mesenchymal stem cells are derived from bone marrow fluid.
8). 8. The cell culture method according to any one of 5 to 7, wherein the medium further contains IL-8 and / or MIP-1α.
１．細胞を増殖培養するための培地であって、ＭＩＧ及びＩ－３０９を含有する、細胞培養用培地。
２．前記細胞が間葉系幹細胞である、前記１に記載の細胞培養用培地。
３．前記間葉系幹細胞が骨髄液由来である、前記２に記載の細胞培養用培地。
４．さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、前記１～３のいずれか１に記載の細胞培養用培地。
５．細胞を、ＭＩＧ及びＩ－３０９を含有する培地で増殖培養する、細胞培養方法。
６．前記細胞が間葉系幹細胞である、前記５に記載の細胞培養方法。
７．前記間葉系幹細胞が骨髄液由来である、前記６に記載の細胞培養方法。
８．前記培地が、さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、前記５～７のいずれか１に記載の細胞培養方法。 That is, the present invention is as follows.
1. A medium for cell culture, which contains MIG and I-309.
2. 2. The cell culture medium according to 1 above, wherein the cells are mesenchymal stem cells.
3. 3. The cell culture medium according to 2 above, wherein the mesenchymal stem cells are derived from bone marrow fluid.
4). 4. The cell culture medium according to any one of 1 to 3, further comprising IL-8 and / or MIP-1α.
5). A cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
6). 6. The cell culture method according to 5 above, wherein the cell is a mesenchymal stem cell.
7). 7. The cell culture method according to 6 above, wherein the mesenchymal stem cells are derived from bone marrow fluid.
8). 8. The cell culture method according to any one of 5 to 7, wherein the medium further contains IL-8 and / or MIP-1α.
特定のサイトカインを含有する本発明の培地を用いることによって、少量の細胞であってもより短時間にかつ多くの細胞を増殖させることができる。特に、患者等の体内から採取した細胞を培養し、培養した細胞を再度患者の体内へ戻す治療を行うような細胞医療等の場合には、患者から採取する細胞の量を少なくすることができるので、患者の負担を軽減して治療を行うことができる。
By using the medium of the present invention containing a specific cytokine, a large number of cells can be proliferated in a shorter time even with a small amount of cells. In particular, in the case of cell therapy or the like in which cells collected from the body of a patient are cultured and the cultured cells are returned to the patient's body, the amount of cells collected from the patient can be reduced. Therefore, treatment can be performed while reducing the burden on the patient.
以下に、本発明を実施するための形態について詳細に説明する。
Hereinafter, embodiments for carrying out the present invention will be described in detail.
［細胞培養用培地］
本発明は、細胞を増殖培養するための培地であって、ＭＩＧ及びＩ－３０９を含有する、細胞培養用培地を提供するものである。 [Cell culture medium]
The present invention provides a medium for cell culture, which is a medium for cell culture and contains MIG and I-309.
本発明は、細胞を増殖培養するための培地であって、ＭＩＧ及びＩ－３０９を含有する、細胞培養用培地を提供するものである。 [Cell culture medium]
The present invention provides a medium for cell culture, which is a medium for cell culture and contains MIG and I-309.
本発明の細胞培養用培地に用いることのできる基礎培地としては、特に限定されないが、通常細胞を増殖するに際して必須成分である、アミノ酸、ビタミン類、無機塩類などを含む。基礎培地としては、例えばイーグル基本培地（ＭＥＭ）、アルファイーグル基本培地（αＭＥＭ）、ダルベッコ改変イーグル培地（ＤＭＥＭ）、ハムＦ－１２培地などが挙げられる。
The basal medium that can be used for the cell culture medium of the present invention is not particularly limited, but includes amino acids, vitamins, inorganic salts, and the like, which are essential components for normal cell growth. Examples of the basal medium include Eagle basal medium (MEM), Alpha Eagle basal medium (αMEM), Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium.
なお、本明細書における「基礎培地」とは、本発明の細胞培養用培地においてＭＩＧ及びＩ－３０９等が添加される前の培地を指し、上記のような市販の基本培地等が該当する。
In addition, the “basal medium” in the present specification refers to a medium before MIG, I-309 and the like are added to the cell culture medium of the present invention, and corresponds to the above-described commercially available basic medium and the like.
本発明の細胞培養用培地は、少なくともＭＩＧ及びＩ－３０９を含有する。
The cell culture medium of the present invention contains at least MIG and I-309.
ＭＩＧはタンパク質で、サイトカインの１種であり、ＣＸＣＬ９とも呼ばれるＣＸＣケモカインファミリーの一つである。ＭＩＧは、インターフェロンγの刺激を受けた単球、マクロファージや内皮細胞から産生され、Ｔｈ１リンパ球の走化性を示すことが知られている。また、腫瘍細胞増殖、血管新生、造血幹前駆細胞のコロニー形成を阻害するともいわれている。本発明においては、例えば和光純薬社製のＭＩＧを用いることができる。
MIG is a protein, a kind of cytokine, and one of the CXC chemokine family also called CXCL9. MIG is known to be produced from monocytes, macrophages, and endothelial cells stimulated with interferon γ and exhibit chemotaxis of Th1 lymphocytes. It is also said to inhibit tumor cell growth, angiogenesis, and hematopoietic stem progenitor cell colony formation. In the present invention, for example, MIG manufactured by Wako Pure Chemical Industries, Ltd. can be used.
Ｉ－３０９はタンパク質で、サイトカインの１種であり、ＣＣＬ１とも呼ばれるＣＣケモカインファミリーの一つである。Ｔリンパ球、単球やマスト細胞で産生され、好中球、マクロファージ、血管平滑筋細胞を誘導し、一部の細胞の増殖を促進するといわれている。本発明においては、例えば和光純薬社製のＩ－３０９を用いることができる。
I-309 is a protein, a kind of cytokine, and is one of the CC chemokine family also called CCL1. Produced by T lymphocytes, monocytes and mast cells, it is said to induce neutrophils, macrophages and vascular smooth muscle cells and promote the proliferation of some cells. In the present invention, for example, I-309 manufactured by Wako Pure Chemical Industries, Ltd. can be used.
本発明の細胞培養用培地は、培地全体に対して、好ましくはＭＩＧを１００～１０００ｎｇ／ｍＬ含有し、より好ましくは３７５～５００ｎｇ／ｍＬ含有する。１０００ｎｇ／ｍＬを超えると、培養コストの増加というおそれがある。
The cell culture medium of the present invention preferably contains 100 to 1000 ng / mL, more preferably 375 to 500 ng / mL of MIG with respect to the entire medium. If it exceeds 1000 ng / mL, the culture cost may increase.
本発明の細胞培養用培地は、培地全体に対して、好ましくはＩ－３０９を５０～１０００ｎｇ／ｍＬ含有し、より好ましくは１００～５００ｎｇ／ｍＬ含有する。１０００ｎｇ／ｍＬを超えると、培養コストの増加というおそれがある。
The cell culture medium of the present invention preferably contains 50 to 1000 ng / mL, more preferably 100 to 500 ng / mL of I-309 with respect to the whole medium. If it exceeds 1000 ng / mL, the culture cost may increase.
また、本発明の細胞培養用培地は、ＭＩＧとＩ－３０９とを、質量比で、１：０．０５～１：１で含有することが好ましく、１：０．０５～１：０．１５で含有することがより好ましい。前記範囲であることによって、より高い細胞増殖促進効果を得られる。
The cell culture medium of the present invention preferably contains MIG and I-309 at a mass ratio of 1: 0.05 to 1: 1, and 1: 0.05 to 1: 0.15. It is more preferable to contain. By being in the above range, a higher cell proliferation promoting effect can be obtained.
本発明の細胞培養用培地は、他のサイトカインとして、さらにＩＬ－８及び／又はＭＩＰ－１αを含有していてもよい。ＩＬ－８及びＭＩＰ－１αもサイトカインの一種であるケモカインに分類され、さらにＩＬ－８はＣＸＣケモカインに、ＭＩＰ－１αはＣＣケモカインに分類される。本発明においては、例えば和光純薬社製のＩＬ－８およびＭＩＰ－１αを用いることができる。
The cell culture medium of the present invention may further contain IL-8 and / or MIP-1α as another cytokine. IL-8 and MIP-1α are also classified as chemokines, which are a kind of cytokine, and IL-8 is classified as a CXC chemokine and MIP-1α is classified as a CC chemokine. In the present invention, for example, IL-8 and MIP-1α manufactured by Wako Pure Chemical Industries, Ltd. can be used.
本発明の細胞培養用培地は、上記以外のその他のサイトカインも、培養する細胞の種類や目的に応じて、本発明の効果を損なわない範囲で任意の成分を含有していてもよい。
The cell culture medium of the present invention may also contain other components, in addition to the above, depending on the type and purpose of the cells to be cultured as long as the effects of the present invention are not impaired.
その他のサイトカインとしては、例えば、ＭＣＰ３、ＲＡＮＴＥＳ、ＭＩＰ－１β、ＩＬ－６、ＩＬ－１０、ＭＣＰ２、ＣＣＬ２４、ＧＲＯ、ｕＰＡＲ、Ｍ－ＣＳＦ、ＮＡＰ－２、ｓｇｐ１３０、ＴＩＭＰ１、ＥＮＡ－７８、ＯＰＧ、ＩＧＦＢＰ２、ＡＮＧ、ＩＬ－７、ＶＥＧＦ－Ａ等を挙げることができる。
Examples of other cytokines include, for example, MCP3, RANTES, MIP-1β, IL-6, IL-10, MCP2, CCL24, GRO, uPAR, M-CSF, NAP-2, sgp130, TIMP1, ENA-78, OPG, Examples include IGFBP2, ANG, IL-7, and VEGF-A.
本発明の細胞培養用培地は、本発明の効果を損なわない範囲で、培養する細胞の種類や目的に応じて、栄養成分、血清、抗生物質等を含有してもよい。
The cell culture medium of the present invention may contain nutrients, serum, antibiotics, and the like depending on the type and purpose of the cells to be cultured within a range that does not impair the effects of the present invention.
栄養成分としては、例えば、脂肪酸等、ビタミン等を挙げることができる。
Examples of nutritional components include fatty acids and vitamins.
血清としては、異種血清および同種血清を用いることができる。異種血清は、細胞培養物を細胞医療等に用いる場合、そのレシピエントとは異なる種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ウシやウマに由来する血清、例えば、ウシ胎仔血清（ＦＢＳ、ＦＣＳ）、仔ウシ血清（ＣＳ）、ウマ血清（ＨＳ）などが異種血清に該当する。また、「同種血清」は、レシピエントと同一の種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ヒト血清が同種血清に該当する。同種血清は、自己血清（自家血清ともいう）、すなわち、レシピエントに由来する血清、およびレシピエント以外の同種個体に由来する同種他家血清を含む。
As the serum, heterologous serum and allogeneic serum can be used. The heterologous serum means serum derived from a different species of organism from the recipient when the cell culture is used for cell medicine or the like. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum. “Allogeneic serum” means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autoserum (also called autologous serum), ie, serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient.
抗生物質としては、例えば、ペニシリン、ストレプトマイシン、ゲンタマイシン等が挙げられる。
Examples of antibiotics include penicillin, streptomycin, gentamicin and the like.
本発明の培地で培養する細胞の動物種は特に制限されず、該細胞を使用する用途に応じて、例えば、ヒト、ラット、マウス、ブタ等を使用できる。
The animal species of the cells cultured in the medium of the present invention is not particularly limited, and for example, humans, rats, mice, pigs and the like can be used according to the use of the cells.
本発明の培地で培養する細胞の種類は、特に限定されるものではないが、例えば、外胚葉系細胞、中胚葉系細胞、内胚葉系細胞、受精卵からこれらの細胞へ分化する過程に含まれる細胞、胚性幹細胞および体性幹細胞等を挙げることができる。
The type of cells to be cultured in the medium of the present invention is not particularly limited. For example, it is included in the process of differentiation from ectodermal cells, mesoderm cells, endoderm cells, and fertilized eggs into these cells. Cells, embryonic stem cells and somatic stem cells.
外胚葉系細胞としては、例えば、ニューロン細胞、アストロサイト細胞、オリゴデンドロサイト細胞およびこれらの幹細胞である神経幹細胞等を挙げることができる。
Examples of ectoderm cells include neuronal cells, astrocyte cells, oligodendrocyte cells, and neural stem cells that are these stem cells.
中胚葉系細胞としては、例えば、血管細胞、造血系細胞、間葉系細胞およびこれらの幹細胞等を挙げることができる。
Examples of mesoderm cells include vascular cells, hematopoietic cells, mesenchymal cells, and stem cells thereof.
造血系細胞としては、例えば、造血幹細胞、造血前駆細胞、赤血球細胞、リンパ球細胞、顆粒球細胞および血小板細胞等を挙げることができる。
Examples of hematopoietic cells include hematopoietic stem cells, hematopoietic progenitor cells, red blood cells, lymphocyte cells, granulocyte cells, and platelet cells.
間葉系細胞としては、例えば、骨細胞、軟骨細胞、筋細胞、心筋細胞、腱細胞、脂肪細胞、毛乳頭細胞、歯髄細胞およびこれらの幹細胞である間葉系幹細胞等を挙げることができる。間葉系幹細胞としては、例えば、骨髄液、脂肪組織、胎盤組織、臍帯組織、又は歯髄等に含まれるものを使用でき、好ましくは骨髄液由来の間葉系幹細胞である。
Examples of mesenchymal cells include bone cells, chondrocytes, muscle cells, cardiomyocytes, tendon cells, adipocytes, dermal papilla cells, dental pulp cells, and mesenchymal stem cells that are these stem cells. As mesenchymal stem cells, for example, those contained in bone marrow fluid, adipose tissue, placenta tissue, umbilical cord tissue, dental pulp, etc. can be used, and preferably mesenchymal stem cells derived from bone marrow fluid.
内胚葉系細胞としては、例えば、肝細胞、膵外分泌細胞、膵内分泌細胞、胆のう細胞およびこれらの幹細胞等を挙げることができる。
Examples of endoderm cells include hepatocytes, pancreatic exocrine cells, pancreatic endocrine cells, gallbladder cells, and stem cells thereof.
本発明の培地を用いて再生医療や細胞療法等に用いる細胞を培養する場合、細胞として好ましくは、幹細胞、より好ましくは間葉系幹細胞が挙げられる。間葉系幹細胞は、例えば、骨髄液由来、脂肪組織由来、又は臍帯血由来等のいずれの組織由来であってもよいが、本発明の効果の観点や、より多くの細胞を採取できるという観点から、好ましくは骨髄液由来の間葉系幹細胞である。
When culturing cells used for regenerative medicine or cell therapy using the medium of the present invention, the cells are preferably stem cells, more preferably mesenchymal stem cells. The mesenchymal stem cell may be derived from any tissue such as bone marrow fluid, adipose tissue, or umbilical cord blood, for example, from the viewpoint of the effect of the present invention and from the viewpoint that more cells can be collected. Therefore, it is preferably a mesenchymal stem cell derived from bone marrow fluid.
［細胞培養方法］
本発明は、細胞を、ＭＩＧ及びＩ－３０９を含有する培地で増殖培養する、細胞培養方法を提供するものである。 [Cell culture method]
The present invention provides a cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
本発明は、細胞を、ＭＩＧ及びＩ－３０９を含有する培地で増殖培養する、細胞培養方法を提供するものである。 [Cell culture method]
The present invention provides a cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
本発明の細胞培養方法は、ＭＩＧ及びＩ－３０９を含有する培地、すなわち上述した本発明の培地を用いて行う。
The cell culture method of the present invention is performed using a medium containing MIG and I-309, that is, the above-described medium of the present invention.
以下、本発明の細胞培養方法において、培養する細胞を間葉系幹細胞として説明をするが、本発明の細胞培養方法は間葉系幹細胞に限定されるものではない。細胞の培養条件等は、細胞の種類等によって適宜好ましい条件を採用できる。
Hereinafter, in the cell culture method of the present invention, the cells to be cultured are described as mesenchymal stem cells, but the cell culture method of the present invention is not limited to mesenchymal stem cells. As cell culture conditions and the like, preferable conditions can be appropriately adopted depending on the type of cells.
間葉系幹細胞の入手方法は、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、マウス等の固体から単離することにより入手する方法、すでにクローン化された各細胞を、各種機関から入手する方法などが挙げられる。
The method for obtaining mesenchymal stem cells is not particularly limited and can be appropriately selected according to the purpose. For example, a method obtained by isolating from a solid such as human or mouse, each cell already cloned. Can be obtained from various institutions.
また、本発明の培地を用いて、細胞を培養し、種々の疾患患者の体内へ戻す治療を行う場合は、該患者の体内から採取した細胞を用いることが好ましい。例えば、肝疾患の患者の治療を行うために、間葉系幹細胞を当該患者に投与する場合は、当該患者の骨髄液の間葉系幹細胞を含む対象液を少量採取して、本発明の培地で培養し、細胞の増殖を行うことが好ましい。
In addition, when a cell is cultured using the medium of the present invention and returned to the body of patients with various diseases, it is preferable to use cells collected from the body of the patient. For example, when mesenchymal stem cells are administered to a patient to treat a patient with liver disease, a small amount of a target solution containing mesenchymal stem cells of the bone marrow of the patient is collected, and the medium of the present invention It is preferable to perform cell proliferation by culturing in
まず、患者から骨髄液を少量（１０～１００ｍＬ）採取する。次にその骨髄液から間葉系幹細胞を分離する。骨髄液から間葉系幹細胞を分離する方法は、従来知られた種々の方法を採用できる。
First, collect a small amount (10-100 mL) of bone marrow from the patient. Next, the mesenchymal stem cells are separated from the bone marrow fluid. As a method for separating mesenchymal stem cells from bone marrow fluid, various conventionally known methods can be adopted.
具体的には、例えば、骨髄液を分注したチューブにＰＢＳ（リン酸緩衝生理食塩水）、ＨＥＳ（赤血球沈降剤）等を使用して赤血球を沈降させる。その後上澄みを抽出し、遠心分離することにより間葉系幹細胞を含む対象液を分離することができる。これらの操作は無菌環境に維持されたアイソレータ内や安全キャビネット内で行われる。さらに、対象液を、培地を入れたフラスコ等に播種し、接着した細胞のみを継代・培養していくという方法を用いることができる。
Specifically, for example, erythrocytes are precipitated using PBS (phosphate buffered saline), HES (erythrocyte sedimentation agent) or the like in a tube into which bone marrow fluid has been dispensed. Thereafter, the supernatant is extracted and centrifuged to separate the target liquid containing mesenchymal stem cells. These operations are performed in an isolator or a safety cabinet maintained in a sterile environment. Furthermore, it is possible to use a method in which the target solution is seeded in a flask or the like containing a medium, and only the adhered cells are subcultured and cultured.
間葉系幹細胞を培養する場合は、インキュベータにより例えば、温度３７℃、５％ＣＯ２の条件下で培養する。培養時間に関しては、コンフルエント状態になる前に継代することが好ましい。間葉系幹細胞の継代数としては、特に制限はなく、目的に応じて適宜選択することができる。
When culturing mesenchymal stem cells, it is cultured in an incubator, for example, under conditions of a temperature of 37 ° C. and 5% CO 2 . As for the culture time, it is preferable to subculture before becoming confluent. The number of passages of mesenchymal stem cells is not particularly limited and can be appropriately selected depending on the purpose.
本発明の細胞培養方法により得られる細胞培養液、または該細胞培養液から細胞のみを分離して得られる細胞画分は、例えば、細胞の種類によって種々の細胞医療等に用いることができる。例えば間葉系幹細胞の場合、上述したように肝疾患の患者の治療に用いることができる。
The cell culture solution obtained by the cell culture method of the present invention or the cell fraction obtained by separating only cells from the cell culture solution can be used for various cell medicines, for example, depending on the cell type. For example, in the case of mesenchymal stem cells, as described above, it can be used for the treatment of patients with liver diseases.
［試験例１］
サイトカインの同定（１）
試験例１では、ヒト全骨髄細胞を培養した培養液に含まれるサイトカインの種類を同定すべく、以下のようにヒト全骨髄細胞の培養を行い、得られた培養液の上清に対しサイトカインアレイ分析を行った。 [Test Example 1]
Identification of cytokines (1)
In Test Example 1, in order to identify the type of cytokine contained in the culture medium in which human whole bone marrow cells were cultured, human whole bone marrow cells were cultured as follows, and a cytokine array was obtained from the obtained culture medium supernatant. Analysis was carried out.
サイトカインの同定（１）
試験例１では、ヒト全骨髄細胞を培養した培養液に含まれるサイトカインの種類を同定すべく、以下のようにヒト全骨髄細胞の培養を行い、得られた培養液の上清に対しサイトカインアレイ分析を行った。 [Test Example 1]
Identification of cytokines (1)
In Test Example 1, in order to identify the type of cytokine contained in the culture medium in which human whole bone marrow cells were cultured, human whole bone marrow cells were cultured as follows, and a cytokine array was obtained from the obtained culture medium supernatant. Analysis was carried out.
〈ヒト全骨髄細胞の培養〉
非接着細胞である血球系の細胞と接着細胞である間葉系幹細胞を含むヒト全骨髄細胞の培養を行うため、非接着細胞と接着細胞を同時に培養することのできる培養皿（以下、培養皿Ａと表記する）を使用した。培養皿Ａとしてはナノサイズの高分子ポリマーとクレイで培養皿表面をコーティングしたものを使用した。このように培養皿をコーティングすることにより、培養面の細胞接着力が増加する等により、通常の培養皿では接着しない細胞も接着するため、血球系の細胞と間葉系幹細胞を同時に培養することができる。 <Culture of human whole bone marrow cells>
In order to culture human whole bone marrow cells including hematopoietic cells that are non-adherent cells and mesenchymal stem cells that are adherent cells, a culture dish that can simultaneously culture non-adherent cells and adherent cells (hereinafter, culture dish) A). As the culture dish A, a culture dish surface coated with nano-sized polymer and clay was used. By coating the culture dish in this way, cells that do not adhere in normal culture dishes can be adhered, for example, by increasing the cell adhesion of the culture surface. Therefore, culturing blood cells and mesenchymal stem cells simultaneously Can do.
非接着細胞である血球系の細胞と接着細胞である間葉系幹細胞を含むヒト全骨髄細胞の培養を行うため、非接着細胞と接着細胞を同時に培養することのできる培養皿（以下、培養皿Ａと表記する）を使用した。培養皿Ａとしてはナノサイズの高分子ポリマーとクレイで培養皿表面をコーティングしたものを使用した。このように培養皿をコーティングすることにより、培養面の細胞接着力が増加する等により、通常の培養皿では接着しない細胞も接着するため、血球系の細胞と間葉系幹細胞を同時に培養することができる。 <Culture of human whole bone marrow cells>
In order to culture human whole bone marrow cells including hematopoietic cells that are non-adherent cells and mesenchymal stem cells that are adherent cells, a culture dish that can simultaneously culture non-adherent cells and adherent cells (hereinafter, culture dish) A). As the culture dish A, a culture dish surface coated with nano-sized polymer and clay was used. By coating the culture dish in this way, cells that do not adhere in normal culture dishes can be adhered, for example, by increasing the cell adhesion of the culture surface. Therefore, culturing blood cells and mesenchymal stem cells simultaneously Can do.
この培養皿Ａにダルベッコ改変イーグル培地（ＤＭＥＭ）、ウシ胎仔血清、ゲンタマイシンを添加した培地（以下、培地Ａと表記する）を用いて、ヒト全骨髄細胞（Ｌｏｎｚａ社製 ＢＭＳＣ、２Ｍ－１２５Ｄ）を温度３７℃、５％ＣＯ２の条件下で培養を行った。
また、コントロールとして、上記コーティングを行っていない培養皿（ＩＷＡＫＩティッシュカルチャディッシュ３０００－０３５：培養皿Ｂ）にダルベッコ改変イーグル培地（ＤＭＥＭ）、ウシ胎仔血清、ゲンタマイシンを添加したもの（以下、培地Ｂと表記する）を使用して、上記と同条件にてヒト全骨髄細胞の培養を行った。 Using this culture dish A with Dulbecco's modified Eagle medium (DMEM), fetal bovine serum, and gentamicin (hereinafter referred to as medium A), human whole bone marrow cells (Lonza BMSC, 2M-125D) were prepared. Culturing was performed under conditions of a temperature of 37 ° C. and 5% CO 2 .
In addition, as a control, a culture dish (IWAKI tissue culture dish 3000-035: culture dish B) not coated with the above was added Dulbecco's modified Eagle medium (DMEM), fetal bovine serum, and gentamicin (hereinafter referred to as medium B). The whole human bone marrow cells were cultured under the same conditions as described above.
また、コントロールとして、上記コーティングを行っていない培養皿（ＩＷＡＫＩティッシュカルチャディッシュ３０００－０３５：培養皿Ｂ）にダルベッコ改変イーグル培地（ＤＭＥＭ）、ウシ胎仔血清、ゲンタマイシンを添加したもの（以下、培地Ｂと表記する）を使用して、上記と同条件にてヒト全骨髄細胞の培養を行った。 Using this culture dish A with Dulbecco's modified Eagle medium (DMEM), fetal bovine serum, and gentamicin (hereinafter referred to as medium A), human whole bone marrow cells (Lonza BMSC, 2M-125D) were prepared. Culturing was performed under conditions of a temperature of 37 ° C. and 5% CO 2 .
In addition, as a control, a culture dish (IWAKI tissue culture dish 3000-035: culture dish B) not coated with the above was added Dulbecco's modified Eagle medium (DMEM), fetal bovine serum, and gentamicin (hereinafter referred to as medium B). The whole human bone marrow cells were cultured under the same conditions as described above.
〈培養上清を用いた細胞の培養〉
上記ヒト全骨髄細胞を培養した培地Ａについて、２０００Ｇで２０分間の遠心分離を行い、培養上清を回収した（以下、上清Ａと表記する）。コントロール培地である培地Ｂについても同様の操作を行い、培養上清を回収した（以下、上清Ｂと表記する）。 <Culture of cells using culture supernatant>
About the culture medium A which culture | cultivated the said human whole bone marrow cell, it centrifuged at 2000 G for 20 minutes, and culture | cultivation supernatants were collect | recovered (henceforth supernatant A). The same operation was performed for medium B as a control medium, and the culture supernatant was collected (hereinafter referred to as supernatant B).
上記ヒト全骨髄細胞を培養した培地Ａについて、２０００Ｇで２０分間の遠心分離を行い、培養上清を回収した（以下、上清Ａと表記する）。コントロール培地である培地Ｂについても同様の操作を行い、培養上清を回収した（以下、上清Ｂと表記する）。 <Culture of cells using culture supernatant>
About the culture medium A which culture | cultivated the said human whole bone marrow cell, it centrifuged at 2000 G for 20 minutes, and culture | cultivation supernatants were collect | recovered (henceforth supernatant A). The same operation was performed for medium B as a control medium, and the culture supernatant was collected (hereinafter referred to as supernatant B).
ヒト全骨髄細胞（Ｐ２：第２継代細胞、Ｌｏｎｚａ社製、２Ｍ－１２５Ｄ）を２４ｗｅｌｌ－ｐｌａｔｅ（ｎ＝１０ｅａｃｈ）、５×１０３細胞／ｗｅｌｌの密度で播種し、そこへ上清Ａと上清Ｂを、それぞれ異なるｗｅｌｌに添加し、５％ＣＯ２、３７℃で４日間培養を行った後、ＭＴＳ ａｓｓａｙを行った。ＭＴＳ ａｓｓａｙは、各Ｗｅｌｌの培地除去後、ＣｅｌｌＴｉｔｅｒ ９６Ｒ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社）と基礎培地を質量比で、１：５で調整した試薬を各Ｗｅｌｌに５００ｕｌずつ加え、５％ＣＯ２、３７℃で４０分インキュベートし、４９０ｎｍの吸光度をＩｎｆｉｎｉｔｅ Ｍ２００（ＴＥＣＡＮ社）を用いて測定することにより行った。その結果を図１に示す。図１の結果からわかるように、上清Ａを添加して培養した細胞の方が、上清Ｂを添加して培養した細胞と比較して、細胞増殖性が高かった。
Human whole bone marrow cells (P2: second passage cell, manufactured by Lonza, 2M-125D) were seeded at a density of 24 well-plate (n = 10each), 5 × 10 3 cells / well, and supernatant A and Supernatant B was added to different wells and cultured at 5% CO 2 and 37 ° C. for 4 days, followed by MTS assay. After MTS assay, the medium is removed of the Well, with CellTiter 96 R AQueous One Solution Cell Proliferation Assay (Promega Corp.) and the mass ratio of the basal medium, 1: added in 500ul of reagent prepared in the Well at 5, 5% CO 2. It was incubated at 37 ° C. for 40 minutes, and the absorbance at 490 nm was measured by using Infinite M200 (TECAN). The result is shown in FIG. As can be seen from the results in FIG. 1, the cells proliferated with the addition of the supernatant A were higher in cell proliferation than the cells cultured with the addition of the supernatant B.
〈サイトカインアレイ分析〉
上記上清Ａ及び上清Ｂについて、ＲａｙＢｉｏ Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ Ａｎｔｉｂｏｄｙ Ａｒｒａｙ Ｇ Ｓｅｒｉｅｓ １０００によりサイトカインアレイ分析を行った。その結果、細胞の増殖促進効果の高かった上清Ａには、上清Ｂと比較して、表１に示す１９種類のサイトカインが高濃度に含まれることが分かった。 <Cytokine array analysis>
The supernatant A and the supernatant B were subjected to cytokine array analysis using RayBio Human Cytokine Antibody Array G Series 1000. As a result, it was found that the supernatant A, which had a high cell growth promoting effect, contained 19 kinds of cytokines shown in Table 1 at a higher concentration than the supernatant B.
上記上清Ａ及び上清Ｂについて、ＲａｙＢｉｏ Ｈｕｍａｎ Ｃｙｔｏｋｉｎｅ Ａｎｔｉｂｏｄｙ Ａｒｒａｙ Ｇ Ｓｅｒｉｅｓ １０００によりサイトカインアレイ分析を行った。その結果、細胞の増殖促進効果の高かった上清Ａには、上清Ｂと比較して、表１に示す１９種類のサイトカインが高濃度に含まれることが分かった。 <Cytokine array analysis>
The supernatant A and the supernatant B were subjected to cytokine array analysis using RayBio Human Cytokine Antibody Array G Series 1000. As a result, it was found that the supernatant A, which had a high cell growth promoting effect, contained 19 kinds of cytokines shown in Table 1 at a higher concentration than the supernatant B.
［試験例２］
サイトカインの同定（２）
試験例２では、試験例１で同定した１９種類のサイトカインのうち、細胞の増殖促進効果を有するサイトカインを同定する試験を行った。 [Test Example 2]
Identification of cytokines (2)
In Test Example 2, a test was performed to identify cytokines having a cell growth promoting effect among the 19 types of cytokines identified in Test Example 1.
サイトカインの同定（２）
試験例２では、試験例１で同定した１９種類のサイトカインのうち、細胞の増殖促進効果を有するサイトカインを同定する試験を行った。 [Test Example 2]
Identification of cytokines (2)
In Test Example 2, a test was performed to identify cytokines having a cell growth promoting effect among the 19 types of cytokines identified in Test Example 1.
〈培地の作製〉
ＤＭＥＭ＋Ｇｅｎｔａｍｙｃｉｎ＋１０％ＦＢＳ培地（基礎培地）を作製した。この基礎培地は下記表２に示す処方で作製した。 <Preparation of medium>
DMEM + Gentamycin + 10% FBS medium (basal medium) was prepared. This basal medium was prepared according to the formulation shown in Table 2 below.
ＤＭＥＭ＋Ｇｅｎｔａｍｙｃｉｎ＋１０％ＦＢＳ培地（基礎培地）を作製した。この基礎培地は下記表２に示す処方で作製した。 <Preparation of medium>
DMEM + Gentamycin + 10% FBS medium (basal medium) was prepared. This basal medium was prepared according to the formulation shown in Table 2 below.
上記基礎培地に、試験例１で高濃度が観察された全１９種類のサイトカインを下記表３の処方で添加した「サイトカイン添加培地１」を作製し、実施例１の培地とした。
また、上記基礎培地に、全１９種類のサイトカインのうち４種類のサイトカインを下記表４の処方で添加した「サイトカイン添加培地２」を作製し、実施例２の培地とした。
また、上記基礎培地に、全１９種類のサイトカインのうち２種類のサイトカインを下記表５の処方で添加した「サイトカイン添加培地３」を作製し、実施例３の培地とした。 A “cytokine-addedmedium 1” was prepared by adding all 19 types of cytokines whose high concentrations were observed in Test Example 1 to the basal medium according to the formulation shown in Table 3 below.
In addition, a “cytokine-addedmedium 2” was prepared by adding 4 of the 19 types of cytokines to the basal medium according to the formulation shown in Table 4 below, and used as the medium of Example 2.
In addition, a “cytokine-added medium 3” was prepared by adding 2 of the 19 types of cytokines to the basal medium in accordance with the formulation shown in Table 5 below, and used as the medium of Example 3.
また、上記基礎培地に、全１９種類のサイトカインのうち４種類のサイトカインを下記表４の処方で添加した「サイトカイン添加培地２」を作製し、実施例２の培地とした。
また、上記基礎培地に、全１９種類のサイトカインのうち２種類のサイトカインを下記表５の処方で添加した「サイトカイン添加培地３」を作製し、実施例３の培地とした。 A “cytokine-added
In addition, a “cytokine-added
In addition, a “cytokine-added medium 3” was prepared by adding 2 of the 19 types of cytokines to the basal medium in accordance with the formulation shown in Table 5 below, and used as the medium of Example 3.
〈細胞の培養〉
ヒト骨髄間葉系幹細胞（Ｐ４：第４継代細胞、Ｌｏｎｚａ社製、ＰＴ－２５０１）を４８ｗｅｌｌ－ｐｌａｔｅ（ｎ＝３ｅａｃｈ）、６×１０３細胞／ｗｅｌｌの密度で播種した。初日は、サイトカインを添加しない上記基礎培地で、５％ＣＯ２、３７℃で培養し、２日目よりサイトカイン添加培地１～３それぞれに培地交換した。またコントロール群に関しては、初日同様のサイトカインを添加しない基礎培地に培地交換を行った。培地交換をして、５％ＣＯ２、３７℃の条件下で培養を行い、Ｉｎｃｕｃｙｔｅ（登録商標）ＺＯＯＭ生細胞イメージングシステム（Ｅｓｓｅｎ ＢｉｏＳｃｉｅｎｃｅ社製）を用いて、各ｗｅｌｌの底面積に占める細胞の割合を経時的に算出し、５日間培養後ヒト骨髄間葉系幹細胞の増殖曲線をそれぞれ作製した。結果を図２に示す。 <Cell culture>
Human bone marrow mesenchymal stem cells (P4: 4th passage cell, manufactured by Lonza, PT-2501) were seeded at a density of 48 well-plate (n = 3each) and 6 × 10 3 cells / well. On the first day, the cells were cultured in the above-mentioned basal medium without addition of cytokine at 5% CO 2 and 37 ° C., and the medium was changed to each of cytokine-addedmediums 1 to 3 from the second day. As for the control group, the medium was changed to a basal medium to which no cytokine was added on the first day. After changing the medium, the cells were cultured under conditions of 5% CO 2 and 37 ° C., and using the Incubate (registered trademark) ZOOM live cell imaging system (manufactured by Essen BioScience), the cells occupied in the bottom area of each well The ratio was calculated over time, and after culturing for 5 days, proliferation curves of human bone marrow mesenchymal stem cells were prepared. The results are shown in FIG.
ヒト骨髄間葉系幹細胞（Ｐ４：第４継代細胞、Ｌｏｎｚａ社製、ＰＴ－２５０１）を４８ｗｅｌｌ－ｐｌａｔｅ（ｎ＝３ｅａｃｈ）、６×１０３細胞／ｗｅｌｌの密度で播種した。初日は、サイトカインを添加しない上記基礎培地で、５％ＣＯ２、３７℃で培養し、２日目よりサイトカイン添加培地１～３それぞれに培地交換した。またコントロール群に関しては、初日同様のサイトカインを添加しない基礎培地に培地交換を行った。培地交換をして、５％ＣＯ２、３７℃の条件下で培養を行い、Ｉｎｃｕｃｙｔｅ（登録商標）ＺＯＯＭ生細胞イメージングシステム（Ｅｓｓｅｎ ＢｉｏＳｃｉｅｎｃｅ社製）を用いて、各ｗｅｌｌの底面積に占める細胞の割合を経時的に算出し、５日間培養後ヒト骨髄間葉系幹細胞の増殖曲線をそれぞれ作製した。結果を図２に示す。 <Cell culture>
Human bone marrow mesenchymal stem cells (P4: 4th passage cell, manufactured by Lonza, PT-2501) were seeded at a density of 48 well-plate (n = 3each) and 6 × 10 3 cells / well. On the first day, the cells were cultured in the above-mentioned basal medium without addition of cytokine at 5% CO 2 and 37 ° C., and the medium was changed to each of cytokine-added
また、培養後のサイトカイン添加培地１（実施例１）、サイトカイン添加培地３（実施例３）、及びコントロール培地（基礎培地）についてＭＴＳ ａｓｓａｙを行った。ＭＴＳ ａｓｓａｙは、試験例１と同様にして測定を行った。この結果を図３に示す。
Further, MTS assay was performed on the cytokine-added medium 1 (Example 1), the cytokine-added medium 3 (Example 3), and the control medium (basal medium) after culture. MTS assay was measured in the same manner as in Test Example 1. The result is shown in FIG.
この結果、ＭＩＧとＩ－３０９を添加したサイトカイン添加培地３は、全１９種類のサイトカインを添加したサイトカイン添加培地１と同等のヒト骨髄間葉系幹細胞の増殖促進効果が見られた。
また、ＭＩＧとＩ－３０９に加え、ＩＬ－８とＭＩＰ－１αを添加したサイトカイン添加培地２においても、全１９種類のサイトカインを添加したものと同等の増殖促進効果が見られた。 As a result, the cytokine-added medium 3 supplemented with MIG and I-309 showed the same effect of promoting proliferation of human bone marrow mesenchymal stem cells as the cytokine-addedmedium 1 supplemented with all 19 types of cytokines.
Further, in the cytokine-addedmedium 2 to which IL-8 and MIP-1α were added in addition to MIG and I-309, the same growth promoting effect as that obtained by adding all 19 types of cytokines was observed.
また、ＭＩＧとＩ－３０９に加え、ＩＬ－８とＭＩＰ－１αを添加したサイトカイン添加培地２においても、全１９種類のサイトカインを添加したものと同等の増殖促進効果が見られた。 As a result, the cytokine-added medium 3 supplemented with MIG and I-309 showed the same effect of promoting proliferation of human bone marrow mesenchymal stem cells as the cytokine-added
Further, in the cytokine-added
［試験例３］
試験例２の実施例１の培地において、ＭＩＧとＩ－３０９の添加量を０としたことを除いては実施例１と同様に培地を作成し、これを参考例１の培地とした。すなわち、全１９種類のサイトカインからＭＩＧとＩ－３０９の２種類のサイトカインを除いた１７種類のサイトカインを基礎培地に添加した参考例１の培地を作製した。そして、参考例１の培地を用いて試験例２と同様にヒト骨髄間葉系幹細胞の培養を行い、増殖曲線を作製した。また試験例２と同様に、全１９種類のサイトカインを添加した培地（実施例１の培地）と、サイトカインを添加しない培地（コントロール培地）についても同様に試験を行った。結果を図４に示す。 [Test Example 3]
A medium was prepared in the same manner as in Example 1 except that the amount of MIG and I-309 added was 0 in the medium of Example 1 of Test Example 2, and this was used as the medium of Reference Example 1. That is, the medium of Reference Example 1 was prepared by adding 17 types of cytokines obtained by removing 2 types of cytokines, MIG and I-309, from all 19 types of cytokines to the basal medium. Then, human bone marrow mesenchymal stem cells were cultured in the same manner as in Test Example 2 using the medium of Reference Example 1, and a growth curve was prepared. Similarly to Test Example 2, a test was similarly performed on a medium to which all 19 types of cytokines were added (the medium in Example 1) and a medium to which no cytokine was added (control medium). The results are shown in FIG.
試験例２の実施例１の培地において、ＭＩＧとＩ－３０９の添加量を０としたことを除いては実施例１と同様に培地を作成し、これを参考例１の培地とした。すなわち、全１９種類のサイトカインからＭＩＧとＩ－３０９の２種類のサイトカインを除いた１７種類のサイトカインを基礎培地に添加した参考例１の培地を作製した。そして、参考例１の培地を用いて試験例２と同様にヒト骨髄間葉系幹細胞の培養を行い、増殖曲線を作製した。また試験例２と同様に、全１９種類のサイトカインを添加した培地（実施例１の培地）と、サイトカインを添加しない培地（コントロール培地）についても同様に試験を行った。結果を図４に示す。 [Test Example 3]
A medium was prepared in the same manner as in Example 1 except that the amount of MIG and I-309 added was 0 in the medium of Example 1 of Test Example 2, and this was used as the medium of Reference Example 1. That is, the medium of Reference Example 1 was prepared by adding 17 types of cytokines obtained by removing 2 types of cytokines, MIG and I-309, from all 19 types of cytokines to the basal medium. Then, human bone marrow mesenchymal stem cells were cultured in the same manner as in Test Example 2 using the medium of Reference Example 1, and a growth curve was prepared. Similarly to Test Example 2, a test was similarly performed on a medium to which all 19 types of cytokines were added (the medium in Example 1) and a medium to which no cytokine was added (control medium). The results are shown in FIG.
この結果、全１９種類のサイトカインから、ＭＩＧとＩ－３０９を除いた１７種類のサイトカインを添加した培地では、全１９種類のサイトカインを添加した培地と比較して、増殖促進効果が有意に減少した。このことから、ＭＩＧとＩ－３０９には細胞の増殖促進効果があることが確認できた。
As a result, the growth-promoting effect was significantly reduced in the medium in which 17 types of cytokines except MIG and I-309 were added from all 19 types of cytokines, compared to the medium in which all 19 types of cytokines were added. . From this, it was confirmed that MIG and I-309 have an effect of promoting cell proliferation.
［試験例４］分化能の確認
実施例３のサイトカイン添加培地３を用いて、ヒト骨髄間葉系幹細胞（Ｐ３：第３継代細胞、Ｌｏｎｚａ社製、ＰＴ－２５０１）を５％ＣＯ２、３７℃でコンフルエントになるまで培養した。その後培地を、下記表６処方の脂肪細胞の分化誘導培地に交換し、継続して細胞の培養を行った。培養開始から１３日後に培養された細胞をＯｉｌ Ｒｅｄ Ｏ染色し、ヒト骨髄間葉系幹細胞の脂肪細胞への分化能を評価した。Ｏｉｌ Ｒｅｄ Ｏ染色の結果を図５に示す。 [Test Example 4] Confirmation of differentiation potential Using the cytokine-added medium 3 of Example 3, human bone marrow mesenchymal stem cells (P3: 3rd passage cell, manufactured by Lonza, PT-2501) were added with 5% CO 2 , The cells were cultured at 37 ° C. until confluent. Thereafter, the medium was replaced with an adipocyte differentiation induction medium prescribed in Table 6 below, and the cells were continuously cultured. Cells cultured 13 days after the start of culture were stained with Oil Red O, and the ability of human bone marrow mesenchymal stem cells to differentiate into adipocytes was evaluated. The results of Oil Red O staining are shown in FIG.
実施例３のサイトカイン添加培地３を用いて、ヒト骨髄間葉系幹細胞（Ｐ３：第３継代細胞、Ｌｏｎｚａ社製、ＰＴ－２５０１）を５％ＣＯ２、３７℃でコンフルエントになるまで培養した。その後培地を、下記表６処方の脂肪細胞の分化誘導培地に交換し、継続して細胞の培養を行った。培養開始から１３日後に培養された細胞をＯｉｌ Ｒｅｄ Ｏ染色し、ヒト骨髄間葉系幹細胞の脂肪細胞への分化能を評価した。Ｏｉｌ Ｒｅｄ Ｏ染色の結果を図５に示す。 [Test Example 4] Confirmation of differentiation potential Using the cytokine-added medium 3 of Example 3, human bone marrow mesenchymal stem cells (P3: 3rd passage cell, manufactured by Lonza, PT-2501) were added with 5% CO 2 , The cells were cultured at 37 ° C. until confluent. Thereafter, the medium was replaced with an adipocyte differentiation induction medium prescribed in Table 6 below, and the cells were continuously cultured. Cells cultured 13 days after the start of culture were stained with Oil Red O, and the ability of human bone marrow mesenchymal stem cells to differentiate into adipocytes was evaluated. The results of Oil Red O staining are shown in FIG.
図５に示すように、培地中に脂肪細胞が観察されたことにより、ヒト骨髄間葉系幹細胞の脂肪細胞への分化能が確認できた。すなわち、ＭＩＧとＩ－３０９を添加したサイトカイン添加培地３を用いて、ヒト骨髄間葉系幹細胞を培養することによって、分化能を有するヒト骨髄間葉系幹細胞を増殖培養することができることが確認できた。
As shown in FIG. 5, the ability of human bone marrow mesenchymal stem cells to differentiate into adipocytes was confirmed by the observation of adipocytes in the medium. That is, it can be confirmed that human bone marrow mesenchymal stem cells having differentiation potential can be proliferated and cultured by culturing human bone marrow mesenchymal stem cells using cytokine-added medium 3 supplemented with MIG and I-309. It was.
本発明を特定の態様を用いて詳細に説明したが、本発明の意図と範囲を離れることなく様々な変更及び変形が可能であることは、当業者にとって明らかである。なお本出願は、２０１６年８月１２日付で出願された日本特許出願（特願２０１６－１５９０１５）に基づいており、その全体が引用により援用される。
Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. Note that this application is based on a Japanese patent application filed on August 12, 2016 (Japanese Patent Application No. 2016-159015), which is incorporated by reference in its entirety.
Claims (8)
- 細胞を増殖培養するための培地であって、ＭＩＧ及びＩ－３０９を含有する、細胞培養用培地。 A culture medium for cell culture, which contains MIG and I-309.
- 前記細胞が間葉系幹細胞である、請求項１に記載の細胞培養用培地。 The cell culture medium according to claim 1, wherein the cells are mesenchymal stem cells.
- 前記間葉系幹細胞が骨髄液由来である、請求項２に記載の細胞培養用培地。 The cell culture medium according to claim 2, wherein the mesenchymal stem cells are derived from bone marrow fluid.
- さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、請求項１～３のいずれか１項に記載の細胞培養用培地。 The cell culture medium according to any one of claims 1 to 3, further comprising IL-8 and / or MIP-1α.
- 細胞を、ＭＩＧ及びＩ－３０９を含有する培地で増殖培養する、細胞培養方法。 A cell culture method in which cells are grown and cultured in a medium containing MIG and I-309.
- 前記細胞が間葉系幹細胞である、請求項５に記載の細胞培養方法。 The cell culture method according to claim 5, wherein the cells are mesenchymal stem cells.
- 前記間葉系幹細胞が骨髄液由来である、請求項６に記載の細胞培養方法。 The cell culture method according to claim 6, wherein the mesenchymal stem cells are derived from bone marrow fluid.
- 前記培地が、さらにＩＬ－８及び／又はＭＩＰ－１αを含有する、請求項５～７のいずれか１項に記載の細胞培養方法。 The cell culture method according to any one of claims 5 to 7, wherein the medium further contains IL-8 and / or MIP-1α.
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| JP2016-159015 | 2016-08-12 | ||
| JP2016159015A JP2018023343A (en) | 2016-08-12 | 2016-08-12 | Medium for cell culture, and cell culture method |
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| WO2022014693A1 (en) | 2020-07-17 | 2022-01-20 | デクソンファーマシューティカルズ株式会社 | Composition, binding inhibitor, medical device, and method for preventing covid-19 |
| WO2022054565A1 (en) | 2020-09-08 | 2022-03-17 | デクソンファーマシューティカルズ株式会社 | Cytokine storm inhibitor, method for using cytokine storm inhibitor, and method for screening for cytokine inhibitor |
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| JP2008500809A (en) * | 2004-03-09 | 2008-01-17 | ライフスキャン・インコーポレイテッド | Methods for generating insulin producing cells |
| JP2011067175A (en) * | 2009-09-28 | 2011-04-07 | Gc Corp | Method for culturing mesenchymal stem cell |
| JP2013505011A (en) * | 2009-09-18 | 2013-02-14 | フジフィルム・ダイオシンス・バイオテクノロジーズ ・ユーケイ・リミテッド | Stem cell conditioned medium composition |
| JP2016025858A (en) * | 2008-10-08 | 2016-02-12 | イントレキソン コーポレーション | Engineered cells expressing multiple immunomodulators and uses thereof |
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2016
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2017
- 2017-08-09 WO PCT/JP2017/028861 patent/WO2018030448A1/en active Application Filing
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| JP2008500809A (en) * | 2004-03-09 | 2008-01-17 | ライフスキャン・インコーポレイテッド | Methods for generating insulin producing cells |
| JP2016025858A (en) * | 2008-10-08 | 2016-02-12 | イントレキソン コーポレーション | Engineered cells expressing multiple immunomodulators and uses thereof |
| JP2013505011A (en) * | 2009-09-18 | 2013-02-14 | フジフィルム・ダイオシンス・バイオテクノロジーズ ・ユーケイ・リミテッド | Stem cell conditioned medium composition |
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