JP4292032B2 - Method for culturing mesenchymal stem cells - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for culturing mesenchymal stem cells that can be favorably cultured even with a serum-free culture medium, mesenchymal stem cells cultured by the culturing method, and The present invention relates to a cultured tissue using leaf stem cells.
[0002]
[Prior art]
Recent advances in cell engineering technology have enabled the culturing of numerous animal cells, and research on so-called regenerative medicine that uses these cells to reconstruct human tissues and organs is rapidly progressing. . Among them, mesenchymal stem cells (MSC) can differentiate into cells that constitute various tissues such as bone, cartilage, fat, ligament, stroma, skeletal muscle, cardiac muscle, smooth muscle, vascular endothelium, and nerve cells. Therefore, application to regenerative medicine is expected.
[0003]
Such mesenchymal stem cells are usually cultured using a culture solution containing bovine serum (FCS or FBS) at a concentration of about 5 to 10% by weight. Bovine serum is widely used for culturing various cells because it promotes cell growth and also has a protective function for cells. For example, Patent Document 1 describes a method of culturing mesenchymal stem cells using a culture medium containing fetal bovine serum (FBS) in the presence of a basement membrane extracellular matrix. However, there is a problem in using bovine serum having a risk of an infectious disease or the like for culturing cells used for regenerative medicine on the premise of transplantation to humans without needing to see the problem of mad cow disease in recent years. In contrast, Patent Document 1 also describes a culture method using human serum instead of fetal bovine serum (FBS), but there is a limit to the amount of collection when using autologous serum, In addition, when using serum from another family, there is a risk of infection, which is the same as when using bovine serum.
Therefore, a culture method capable of efficiently culturing mesenchymal stem cells using a culture solution that does not contain biological components such as serum has been demanded.
[0004]
[Patent Document 1]
Japanese Patent Laid-Open No. 2003-52360
[Problems to be solved by the invention]
In view of the above situation, the present invention provides a method for culturing mesenchymal stem cells that can be cultured well even with a serum-free culture medium, and mesenchymal stem cells cultured by the culturing method. Another object is to provide a cultured tissue using the mesenchymal stem cells.
[0006]
[Means for Solving the Problems]
The present invention is a method for culturing mesenchymal stem cells using a culture solution containing at least basic fibroblast growth factor (bFGF).
The present invention is described in detail below.
[0007]
In the method for culturing mesenchymal stem cells of the present invention, a culture solution containing at least a basic fibroblast growth factor (hereinafter also referred to as bFGF) is used. As a result of intensive studies, the present inventors have found that mesenchymal stem cells can be cultured by using bFGF in the same manner as in the case of using a serum-containing culture solution even when using a culture solution not containing serum. The headline and the present invention were completed.
[0008]
The above-mentioned bFGF means a non-naturally occurring fibroblast growth factor artificially produced by recombinant DNA or the like.
As the bFGF, a product produced by preparing a recombinant DNA and expressing it may be used, or a commercially available product may be used. Examples of the commercially available bFGF include “Trafermin” (manufactured by Kaken Pharmaceutical Co., Ltd.).
[0009]
The concentration of bFGF in the culture medium is not particularly limited, but is preferably adjusted as appropriate depending on the number of mesenchymal stem cells to be cultured. The preferred lower limit is 0.1 μg / 5 × 10 ⁶ cells, and the preferred upper limit is 1000 μg / 5 × 10 ⁶ cells. When the amount is less than 0.1 μg / 5 × 10 ⁶ cells, the mesenchymal stem cells may not sufficiently proliferate, and when the amount exceeds 1000 μg / 5 × 10 ⁶ cells, the mesenchymal stem cells can be added even if they are further added. The cost of culture may increase without affecting the growth. A more preferable lower limit is 1 μg / 5 × 10 ⁶ cells, and a more preferable upper limit is 100 μg / 5 × 10 ⁶ cells.
[0010]
Components other than bFGF in the culture medium are not particularly limited, and conventionally known basic culture liquids such as Dulbecco's modified Eagle culture liquid (DMEM), ham F12 culture liquid, or a mixture thereof can be used.
[0011]
The culture medium preferably does not contain serum. Mesenchymal stem cells cultured using a culture solution containing serum cannot be used for applications such as regenerative medicine because there is a risk of causing infections and the like. By containing the bFGF, mesenchymal stem cells can be sufficiently cultured without containing serum.
[0012]
The subject of the mesenchymal stem cell culture method of the present invention is not particularly limited as the origin of the mesenchymal stem cell, and mammals such as humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, mice; Those derived from insects and the like can be used. Of these, those derived from humans are preferred for the purpose of regenerative medicine.
The mesenchymal stem cells can be collected from the aforementioned derived organisms by a conventionally known method. For example, it can be obtained by separating cells from bone marrow fluid by density gradient centrifugation and selecting from the separated cells with cell surface markers such as CD14, CD34, CD45.
[0013]
According to the method for culturing mesenchymal stem cells of the present invention, mesenchymal stem cells can be cultured well even in a serum-free system. Since mesenchymal stem cells cultured by the culture method of the present invention have a low risk of infection or the like, they can be suitably used for the purpose of regenerative medicine by preparing a cultured tissue. Furthermore, mesenchymal stem cells cultured by the method of culturing mesenchymal stem cells of the present invention are extremely normal, and if they are seeded on a culture substrate or the like, a cultured tissue is prepared and transplanted into a living body, they are normally differentiated. The target organization can be generated.
The mesenchymal stem cells cultured by the method for culturing mesenchymal stem cells of the present invention are also one aspect of the present invention.
[0014]
The method for preparing a cultured tissue using the mesenchymal stem cells of the present invention is not particularly limited, and the mesenchymal stem cells of the present invention are seeded on a culture substrate, and the culture solution for mesenchymal stem cells of the present invention is used. Examples thereof include a culture method. At this time, a culture solution for differentiating mesenchymal stem cells may be used in accordance with the purpose.
The culture substrate is not particularly limited. For example, natural materials such as collagen, hyaluronic acid, and gelatin; synthetic biodegradable polymer materials such as poly-L-lactic acid, polyglycolic acid, and poly-ε-caprolactone are used. The thing which consists of is mentioned.
The target tissue can be generated by transplanting the cultured tissue thus prepared into a living body. Examples of tissues to be regenerated include skin, skin appendages, bones, cartilage, and muscles.
A cultured tissue using the mesenchymal stem cells of the present invention is also one aspect of the present invention.
[0015]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
[0016]
Example 1
For mesenchymal stem cells by adding bFGF (“Trafermin”, manufactured by Kaken Pharmaceutical Co., Ltd.) to Dulbecco's modified Eagle culture medium (DMEM) (“Invitrogen”, Life Technologies) to a concentration of 2.5 ng / mL A culture solution was prepared.
[0017]
A cell suspension is prepared by suspending human mesenchymal stem cells (manufactured by Biorettacker) in Dulbecco's modified Eagle medium (DMEM), and the concentration is 2.5 × 10 ⁴ cells / plate in a 24-well cell culture plate. And cultured for 24 hours in an environment of 37 ° C. and 5% CO ₂ to adhere the cells to the culture plate.
Subsequently, the culture solution was replaced with 0.5 mL of a mesenchymal stem cell culture solution. This time point was designated as the 0th day of culture. Thereafter, the culture was continued for 3 days, and the number of cells on the culture 1, 2 and 3 days was measured.
The change in the number of cells is shown in FIG.
[0018]
(Control 1)
A culture solution was prepared by adding bovine serum (FBS) to Dulbecco's modified Eagle culture solution (DMEM) (“Invitrogen”, Life Technology) to a concentration of 10% by weight.
The culture was continued for 3 days in the same manner as in Example 1 except that this culture solution was used as the culture solution used on and after the 0th day of culture, and the number of cells on the cultures 1, 2 and 3 was measured.
The change in the number of cells is shown in FIG.
[0019]
(Comparative Example 1)
The culture was continued for 3 days in the same manner as in Example 1 except that Dulbecco's modified eagle culture medium (DMEM) to which neither bFGF nor serum was added was used as the culture medium used after the 0th day of culture. The cell number of the day was measured.
The change in the number of cells is shown in FIG.
[0020]
(Example 2)
A cell suspension is prepared by suspending human mesenchymal stem cells (manufactured by Biorettacker) in Dulbecco's modified Eagle medium (DMEM), seeded at 1 × 10 ⁶ cells in a cell culture dish having a diameter of 100 mm, and 37 ° C. The cells were allowed to adhere to the cell culture dish by culturing for 24 hours in a 5% CO ₂ environment.
The culture solution was replaced with 10 mL of the mesenchymal stem cell culture solution prepared in Example 1, and the culture was continued until subconfluent while changing the culture solution every 3 days. The subconfluent mesenchymal stem cells were treated with 0.25% trypsin / 1 mLEDTA, peeled off from the cell culture dish, recovered, and again seeded at 5 × 10 ⁶ cells in a cell culture dish having a diameter of 100 mm. Mesenchymal stem cells (4 passage cells) obtained by repeating this operation 4 times were used for the production of cultured tissues.
[0021]
The obtained 4 passage cells were suspended in 1 mL of Dulbecco's modified Eagle's culture medium (DMEM) supplemented with 10 μg of bFGF to prepare a cell suspension, and the entire amount was cut into a size of 1.5 × 1.5 cm. The seeds were sown on artificial skin composed of a collagen sponge and a silicone layer (“Pernac”, manufactured by Gunze).
A 1.5 × 1.5 cm full-thickness wound was created on the back of a nude rat (F344 / NJC1-rnu), and artificial skin seeded with the obtained cells was transplanted.
When the wound after transplantation was observed, epidermis was observed on the seventh day of transplantation. Further, 42 days after transplantation, the wound was collected, and the section was observed by staining with hematoxylin-eosin. As a result, it was confirmed that the skin tissue was completely regenerated.
[0022]
【The invention's effect】
According to the present invention, a mesenchymal stem cell culturing method capable of culturing mesenchymal stem cells satisfactorily even using a serum-free medium, a mesenchymal stem cell cultured by the culturing method, and A cultured tissue using the mesenchymal stem cells can be provided.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in the number of cells in culture 1, 2 and 3 days in Example 1, Control Example 1 and Comparative Example 1. FIG.

Claims (1)

A method for culturing mesenchymal stem cells using a culture solution comprising a basic culture solution and basic fibroblast growth factor (bFGF),
A concentration of the basic fibroblast growth factor (bFGF) is 0.1 to 1000 μg / 5 × 10 6. ⁶ cells
A mesenchymal stem cell culture method characterized by the above.

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