WO2018030448A1 - Milieu de culture de cellules et procédé de culture de cellules - Google Patents

Milieu de culture de cellules et procédé de culture de cellules Download PDF

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Publication number
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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Prior art keywords
cells
medium
cell culture
stem cells
mesenchymal stem
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PCT/JP2017/028861
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English (en)
Japanese (ja)
Inventor
功 坂井田
太郎 高見
祐希 相部
健二 米田
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国立大学法人山口大学
澁谷工業株式会社
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Publication of WO2018030448A1 publication Critical patent/WO2018030448A1/fr

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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
    • 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

Definitions

  • 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

L'objet de la présente invention est d'obtenir un milieu contenant une cytokine spécifique et donc pouvant faire croître et cultiver de nombreuses cellules sur une courte période de temps. La présente invention concerne un milieu de culture de cellules pour la prolifération et la culture de cellules, le milieu contenant MIG et I -309.
PCT/JP2017/028861 2016-08-12 2017-08-09 Milieu de culture de cellules et procédé de culture de cellules WO2018030448A1 (fr)

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JP6830286B1 (ja) 2020-06-26 2021-02-17 デクソンファーマシューティカルズ株式会社 精巣機能改善剤および精巣機能改善方法
CN115768871A (zh) 2020-07-17 2023-03-07 Dexon制药株式会社 组合物、结合抑制剂、医疗器械和covid-19的预防方法
CN116171158A (zh) 2020-09-08 2023-05-26 Dexon制药株式会社 细胞因子风暴抑制剂、细胞因子风暴抑制剂的使用方法和筛选方法
JP6974892B1 (ja) 2021-05-27 2021-12-01 デクソンファーマシューティカルズ株式会社 癌悪液質の改善剤および癌悪液質の改善方法
JP7007770B1 (ja) 2021-06-21 2022-02-10 パナジー株式会社 眼症状改善剤および眼症状の改善方法

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