TW201809269A - Cell culture medium and method for culturing cells - Google Patents

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

Cell culture medium and cell culture method

The present invention relates to a cell culture medium and a cell culture method, and more particularly, to a cell culture medium and a cell culture method for mesenchymal stem cells.

In the field of cell medicine, the use of cells to treat various tissues of the human body has attracted much attention. In particular, stem cells have the ability of self-replication and multi-differentiation, and are assumed to be materials for cells or tissues in regenerative medicine.

These stem cells are roughly divided into two types: pluripotent stem cells and tissue stem cells, which are present in various tissues in a living body. For example, mesenchymal stem cells known as tissue stem cells are contained in bone marrow fluid, adipose tissue, placental tissue, umbilical cord tissue, or dental pulp. Hematopoietic stem cells are known to be mainly contained in bone marrow fluid, and also exist in peripheral blood, umbilical cord. In the blood.

In the field of cell medicine, when cells for treatment are administered to a patient, there are methods for administering the cells to the patient without culturing the cells after being taken from a living body, and also after culturing them once in vitro. How to re-administer to patients.

Using the former method, the present inventors have found that mesenchymal stem cells isolated from bone marrow fluid to date are effective for the treatment of patients with liver disease. Specifically, a bone marrow fluid is taken from a patient with liver disease, and a component containing mesenchymal stem cells is isolated therefrom and concentrated to obtain a preparation containing bone marrow cells for liver regeneration. Intravenous administration to this patient can improve liver disease and restore liver function (Patent Document 1).

On the other hand, the amount of stem cells contained in various tissues is very small. For example, the presence of mesenchymal stem cells in bone marrow fluid is about 0.05%. In order to be directly used in the clinic, a large amount of bone marrow fluid must be taken. . Therefore, stem cells taken from living tissues are ideal for subculture and reuse.

In addition, as for the culture of mesenchymal stem cells, it is known to perform primary culture and subculture of the bone marrow fluid to be used to proliferate mesenchymal stem cells to a target amount (Patent Document 2).

[Prior technical literature] [Patent Literature]

Patent Document 1: Japanese Patent Laid-Open No. 2007-89532

Patent Document 2: Japanese Patent Laid-Open No. 2011-67175

Therefore, the present inventors have attempted to proliferate mesenchymal stem cells to a target amount by collecting bone marrow fluid from a patient with liver disease and culturing it. The inventors initially isolated mesenchymal stem cells from the bone marrow fluid taken, and then cultured them using a commercially available medium. However, the whole bone marrow cells in the bone marrow fluid were directly cultured without mesenchymal stem cells being isolated. As a result of the culture, it was surprisingly found that more cells can be cultured in a shorter period of time than when only mesenchymal stem cells to be proliferated are isolated and cultured.

The inventors of the present inventors conducted further research and found that among various proteins contained in a culture solution obtained by culturing whole bone marrow cells, specific cytokines are effective for cell proliferation. That is, it was found that by using The medium can be used to proliferate a larger number of cells in a shorter time even with a small number of cells, thereby completing the present invention.

That is, the present invention is as follows.

A medium for cell culture, which is used for the proliferative culture of cells, and contains MIG and I-309.

2. The cell culture medium according to the above 1, wherein the cells are mesenchymal stem cells.

3. The medium for cell culture according to the above 2, wherein the mesenchymal stem cells are derived from bone marrow fluid.

4. The cell culture medium according to any one of the above 1 to 3, further comprising IL-8 and / or MIP-1α.

5. A cell culture method, which utilizes a medium containing MIG and I-309 for proliferative culture of cells.

6. The cell culture method according to the above 5, wherein the cells are mesenchymal stem cells.

7. The cell culture method according to the above 6, wherein the mesenchymal stem cells are derived from bone marrow fluid.

8. The cell culture method according to any one of 5 to 7, wherein the medium further contains IL-8 and / or MIP-1α.

By using the medium of the present invention containing a specific cytokine, even a small number of cells can proliferate in a shorter time and more cells. In particular, in the case of treating cell-like medicine such as treating cells taken from the body of a patient, and then transferring the cultured cells back to the body of the patient, the number of samples collected from the patient can be reduced. The amount of cells taken can reduce the burden on the patient for treatment.

FIG. 1 is a graph showing the results of a Multiple Target Screening assay (MTS assay) on human whole bone marrow cells cultured in Test Example 1. FIG.

FIG. 2 is a graph showing a proliferation curve of human bone marrow mesenchymal stem cells and the like cultured in the examples.

FIG. 3 is a graph showing the results of MTS assays of human bone marrow mesenchymal stem cells and the like cultured in the examples.

Fig. 4 is a graph showing a proliferation curve of human bone marrow mesenchymal stem cells and the like cultured in a reference example.

FIG. 5 is a photograph showing differentiation of human bone marrow mesenchymal stem cells cultured into adipocytes in the examples.

The embodiments for carrying out the present invention will be described in detail below.

[Cell culture medium]

The invention provides a cell culture medium, which is used for proliferative culture of cells, and contains MIG and I-309.

The basal medium that can be used in the cell culture medium of the present invention is not particularly limited, and includes amino acids, vitamins, inorganic salts, and the like, which are essential components for generally proliferating cells. Examples of the basic medium include Eagle's Basic Medium (MEM), αEagle's Basic Medium (αMEM), and Du's Modified Eagle Medium (DMEM), Ham's F-12 medium, etc.

The "basal medium" in this specification refers to a medium before adding MIG, I-309, and the like to the cell culture medium of the present invention, and it refers to a commercially available basic medium and the like as described above.

The cell culture medium of the present invention contains at least MIG and I-309.

MIG is a protein, which is a kind of cytokine, and is one of the CXC chemokine hormone family also known as CXCL9. It is known that MIG is produced from monocytes, macrophages, or endothelial cells stimulated by interferon gamma, which indicates the chemotaxis of Th1 lymphocytes. It is also known to inhibit tumor cell proliferation, angiogenesis, and formation of hematopoietic stem precursor cells. In the present invention, for example, MIG manufactured by Wako Pure Chemical Industries, Ltd. can be used.

I-309 is a protein, which is one of the cytokines, and is one of the CC chemokine family also known as CCL1. It is known to be produced in T lymphocytes, monocytes or mast cells, induce neutrophils, macrophages, 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.

The cell culture medium of the present invention preferably contains 100 to 1000 ng / mL of MIG, and more preferably contains 375 to 500 ng / mL of the whole culture medium. If it exceeds 1000 ng / mL, the culture cost may increase.

The cell culture medium of the present invention preferably contains 50-1000 ng / mL of I-309, and more preferably 100-500 ng / mL, 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 MIG and I-309 in a mass ratio of 1: 0.05 to 1: 1, and more preferably contains 1: 0.05 to 1: 0.15. 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-1α acts as another cytokine. IL-8 and MIP-1α are also classified as chemokines as cytokines, and IL-8 is classified as CXC chemokine, and MIP-1α is classified as CC chemokine. In the present invention, for example, IL-8 and MlP-1α manufactured by Wako Pure Chemical Industries, Ltd. can be used.

The medium for cell culture of the present invention may contain arbitrary components such as cytokines other than those described above within a range that does not impair the effects of the present invention, depending on the type or purpose of the cells to be cultured.

Examples of other cytokines include 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, etc.

The medium for cell culture of the present invention may contain nutritional components, serum, antibiotics, etc. depending on the type or purpose of the cells to be cultured, as long as the effect of the present invention is not impaired.

Examples of the nutritional component include fatty acids and the like, and vitamins.

As the serum, a heterologous serum and a homologous serum can be used. Heterologous serum refers to serum derived from an organism of a different species than its recipient when a cell culture is used in cell medicine or the like. For example, when the recipient is a human, serum from bovine or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. is equivalent to xenogeneic serum. The term "same serum" means serum derived from an organism of the same species as the recipient. For example, when the recipient is a human, human serum is equivalent to the same kind of serum. Allogeneic sera include self-serum (also known as autologous serum), that is, serum derived from the recipient, and allogeneic serum derived from the same species other than the recipient.

Examples of the antibiotic include penicillin, streptomycin, and gentamicin (Gentamicin), etc.

There is no particular limitation on the animal species of the cells cultured using the culture medium of the present invention. Depending on the application in which the cells are used, for example, humans, rats, mice, and pigs can be used.

The type of the cells cultured in the medium of the present invention is not particularly limited, and examples thereof include ectodermal cells, mesoderm cells, endoderm cells, cells contained in the process of differentiation of self-fertilized eggs to these cells, and embryonic stem cells. And adult stem cells.

Examples of the ectodermal cells include neuronal cells, astrocytes, oligodendritic cells, and neural stem cells as the stem cells.

Examples of mesodermal cells include vascular cells, hematopoietic cells, mesenchymal cells, and stem cells thereof.

Examples of hematopoietic cells include hematopoietic stem cells, hematopoietic precursor cells, red blood cells, lymphocytes, granulocytes, and platelet cells.

Examples of mesenchymal cells include osteocytes, chondrocytes, muscle cells, cardiomyocytes, tendon cells, adipocytes, skin papilla cells, dental pulp cells, and mesenchymal stem cells as the stem cells. As the mesenchymal stem cells, for example, those contained in bone marrow fluid, adipose tissue, placental tissue, umbilical cord tissue, or dental pulp can be used. Preferably, mesenchymal stem cells are derived from mesenchymal stem cells.

Examples of endoderm cells include hepatocytes, pancreatic exocrine cells, pancreatic endocrine cells, gallbladder cells, and stem cells thereof.

When the cells used for regenerative medicine or cell therapy are cultured using the medium of the present invention, the cells are preferably stem cells, and more preferably, mesenchymal stem cells. Mesenchymal stem cells may be derived from bone marrow fluid, for example, From the viewpoint of the effect of the present invention or the viewpoint that more cells can be taken from any tissue such as adipose tissue or umbilical cord blood, it is preferably derived from mesenchymal stem cells.

[Cell culture method]

The invention provides a cell culture method, which uses cells containing MIG and I-309 to perform proliferative culture on cells.

The cell culture method of the present invention is performed using a medium containing MIG and I-309, that is, the above-mentioned medium of the present invention.

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. The culture conditions and the like of the cells can be appropriately and suitably selected according to the type of the cells and the like.

The method for obtaining mesenchymal stem cells is not particularly limited, and may be appropriately selected according to the purpose. For example, methods for obtaining mesenchymal stem cells are obtained by detaching from individuals such as humans and mice, and obtaining selected cells from various institutions. Methods, etc.

In the case where the cells are cultured using the medium of the present invention and then returned to the body of a patient with various diseases, it is preferable to use cells collected from the body of the patient. For example, in the case where mesenchymal stem cells are administered to a patient for the treatment of a liver disease patient, it is preferable to take a small amount of a target fluid containing mesenchymal stem cells of the patient's bone marrow fluid and use the culture medium of the present invention. Cultivate and proliferate cells.

First, a small amount (10-100 mL) of bone marrow fluid was taken from the patient. Mesenchymal stem cells were then isolated from the bone marrow fluid. Various methods for isolating mesenchymal stem cells from bone marrow fluid can be used.

Specifically, for example, a method in which erythrocytes are precipitated in a tube in which bone marrow fluid is dispensed using PBS (phosphate buffered saline), HES (red blood cell precipitating agent), or the like can be used. Thereafter, the supernatant is extracted and centrifuged, whereby a target liquid containing mesenchymal stem cells can be separated. These operations can be performed in an isolator or a safety cabinet maintained in a sterile environment. The target solution is further sown in a flask or the like containing the culture medium, and the adhered cells are continuously subcultured and cultured.

When the mesenchymal stem cells are cultured, they are cultured in an incubator under conditions such as a temperature of 37 ° C and 5% CO ₂ . Regarding the culture time, it is preferable to carry out the subculture before it becomes confluent. The number of generations of mesenchymal stem cells is not particularly limited, and can be appropriately selected according to the purpose.

The cell culture liquid obtained by the cell culture method of the present invention, or a cell portion obtained by separating only cells from the cell culture liquid can be used in various cell medicines and the like according to the type of cells. For example, in the case of mesenchymal stem cells, it can be used for the treatment of patients with liver diseases as described above.

[Example] [Test Example 1] Identification of cytokines (1)

In Test Example 1, the type of cytokine contained in the culture solution obtained by culturing human whole bone marrow cells should be identified, and the culture of human whole bone marrow cells should be performed by the following method. Cytokines array analysis.

<Culture of Human Whole Bone Marrow Cells>

For the purpose of including blood cells as non-adherent cells and as adherent cells Mesenchymal stem cells are cultured in human whole bone marrow cells using a petri dish capable of culturing non-adherent cells and adherent cells (hereinafter referred to as Petri dish A). As the petri dish A, a nano-sized polymer and clay were used to coat the surface of the petri dish. In this way, by coating the culture dish, the cell adhesion on the culture surface is increased, and thereby the non-adhesive cells in the usual culture dish are also adhered, so that the blood cell and mesenchymal stem cells can be cultured at the same time.

A culture medium (DMEM), fetal calf serum, and gentamicin (hereinafter referred to as culture medium A) supplemented with Duchenne's Modified Eagle's Medium (DMEM) was used in the Petri dish A. The temperature was 37 ° C and 5% CO ₂ was used for human Whole bone marrow cells (BMSC, 2M-125D manufactured by Lonza) were cultured. In addition, as a control group, those who had not been coated as described above (1WAKI Tissue Culture Dish 3000-035: Petri dish B) were supplemented with Dubb's Modified Eagle's Medium (DMEM), fetal bovine serum, and gentamicin. (Hereinafter referred to as culture medium B.) Human whole bone marrow cells were cultured under the same conditions as above.

<Culture of cells using culture supernatant>

Regarding the above-mentioned culture medium A for culturing human whole bone marrow cells, centrifugation was performed at 2000 G for 20 minutes, and a culture supernatant (hereinafter referred to as a supernatant A) was recovered. The same operation was performed on the medium B as a control medium, and a culture supernatant (hereinafter referred to as a supernatant B) was recovered.

Human whole bone marrow cells (P2: 2nd generation cells, manufactured by Lonza, 2M-125D) were seeded at a density of 24well-plate (n = 10each) and 5 × 10 ³ cells / well, and were transferred to different wells. Supernatant A and supernatant B were added and cultured at 5% CO ₂ and 37 ° C for 4 days, and then subjected to MTS assay. The MTS assay is performed as follows: After removing the culture medium of each Well, a reagent prepared by preparing CellTiter 96 ^R Aqueous One Solution Cell Proliferation Assay (Promega) and a basic medium at a mass ratio of 1: 5 is added to each Well one by one. 500 ul, incubate at 37 ° C for 40 minutes at 5% CO ₂ , and measure the absorbance at 490 nm using Infinite M200 (TECAN). The results are shown in Fig. 1. According to the results in FIG. 1, it can be seen that the cells cultured by adding the supernatant A have higher cell proliferation than the cells cultured by adding the supernatant B.

<Cytohormone array analysis>

About the above-mentioned supernatant A and supernatant B, cytohormone array analysis was performed by RayBio Human Cytokine Antibody Array G Series 1000. As a result, it was found that, compared with the supernatant B, the cell proliferation-promoting effect was higher. The supernatant A contained the 19 cytokines shown in Table 1 at a high concentration.

[Test Example 2] Identification of cytokines (2)

In Test Example 2, a test was performed to identify a cytokine having a cell proliferation promoting effect among the 19 cytokines identified in Test Example 1.

<Preparation of Culture Medium>

DMEM + Gentamycin + 10% FBS medium (basal medium) was prepared. This basal medium was prepared using the formulation shown in Table 2 below.

To the above-mentioned basic culture medium, all 19 kinds of cytokines with high concentrations observed in Test Example 1 were added in the formulation of Table 3 below, and a "cytokine-added medium 1" was prepared and used as the medium of Example 1. Furthermore, 4 of the 19 cytokines were added to the above-mentioned basal medium in the formulation of Table 4 below to prepare a "cytokine-added medium 2" and used as the medium of Example 2. In addition, two of the 19 cytokines were added to the above-mentioned basal medium in the formulation of Table 5 below to prepare a "cytokine-added medium 3" and used as the medium of Example 3.

<Cell Culture>

Human bone marrow mesenchymal stem cells (P4: 4th generation cells, manufactured by Lonza Corporation, PT-2501) were seeded at a density of 48 well-plate (n = 3each) and 6 × 10 ³ cells / well. On the first day, the above-mentioned basic medium without cytokine was used, and cultured at 5% CO ₂ at 37 ° C. From the second day, the medium was replaced with cytokine-added medium 1-3. Regarding the control group, the same basal medium without the addition of cytokines as in the first day was subjected to medium replacement. After changing the culture medium, the cells were cultured under the conditions of 5% CO ₂ and 37 ° C., and the Incocyte (registered trademark) ZOOM live cell imaging system (manufactured by Essen BioScience) was used to calculate the area of cells over the bottom of each well over time Proportion. After 5 days of culture, the proliferation curves of human bone marrow mesenchymal stem cells were prepared. The results are shown in FIG. 2.

The 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 the culture. The MTS assay was performed in the same manner as in Test Example 1. The results are shown in FIG. 3.

As a result, the growth promoting effect of human bone marrow mesenchymal stem cells equivalent to the cytokine-added medium 3 containing MIG and I-309 was equivalent to the cytokine-added medium 1 containing all 19 cytokines. In addition, in addition to MIG and I-309, IL-8 and MIP-1α-added cytokine-added medium 2 also exhibited a proliferation-promoting effect equivalent to that of all 19 cytokines.

[Test Example 3]

In the medium of Example 1 of Test Example 2, except that the addition amounts of MIG and I-309 were set to 0, a medium was prepared in the same manner as in Example 1, and this was set as the medium of Reference Example 1. That is, 17 kinds of cytokines obtained by removing two kinds of cytokines, MIG and I-309, from all 19 kinds of cytokines were added to a basic medium to prepare a medium of Reference Example 1. Then, the medium of Reference Example 1 was used, and In Example 2, human bone marrow mesenchymal stem cells were cultured in the same manner, and a proliferation curve was prepared. In the same manner as in Test Example 2, the test was also performed on the medium (the medium of Example 1) to which all 19 kinds of cytokines were added and the medium (control medium) to which no cytokines were added. The results are shown in FIG. 4.

As a result, in a medium supplemented with 17 kinds of cytokines obtained by removing MIG and I-309 from all 19 kinds of cytokines, the proliferation-promoting effect was significantly reduced compared with a medium containing all 19 kinds of cytokines. . This confirmed the proliferation promotion effect of cells in MIG and I-309.

[Test Example 4] Confirmation of differentiation function

Use Example was added 3 of the culture medium of cytokine 3, in under 5% CO _2, 37 ℃ for Human bone marrow mesenchymal stem cells (P3: third subculture cells, Lonza Inc., PT-2501) were cultured until confluent. Thereafter, the culture medium was changed to a differentiation induction medium for the adipocytes of the formula shown in Table 6 below, and the cell culture was continued. Thirteen days after the start of culture, the cultured cells were subjected to Oil Red O staining to evaluate the function of human bone marrow mesenchymal stem cells to differentiate into adipocytes. The results of Oil Red O staining are shown in FIG. 5.

As shown in FIG. 5, the function of differentiating human bone marrow mesenchymal stem cells into adipocytes was confirmed by the observation of adipocytes in the culture medium. That is, it was confirmed that human bone marrow cells were treated with cytokine-added medium 3 containing MIG and I-309. Leaf stem cells can be cultured to proliferate and culture human bone marrow mesenchymal stem cells with differentiation function.

The present invention has been described in detail using specific aspects, but practitioners should know that various changes and modifications can be made without departing from the spirit and scope of the present invention. Furthermore, 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)

A cell culture medium is used for the proliferative culture of cells, and contains MIG and I-309. The medium for cell culture according to claim 1, wherein the cells are mesenchymal stem cells. The cell culture medium according to claim 2, wherein the mesenchymal stem cells are derived from bone marrow fluid. The cell culture medium according to any one of claims 1 to 3, further comprising IL-8 and / or MIP-1α. A cell culture method involves proliferating a cell using a medium containing MIG and I-309. The cell culture method according to claim 5, wherein the cells are mesenchymal stem cells. The cell culture method according to claim 6, wherein the mesenchymal stem cells are derived from bone marrow fluid. The cell culture method according to any one of claims 5 to 7, wherein the medium further contains IL-8 and / or MIP-1α.