JPH06169761A - Culture medium for serum-free culture of fibroblast derived from animal and method for culturing - Google Patents

Abstract

(57) [Summary] [Object] To provide a serum-free culture medium and a culture method for obtaining the proliferation of animal-derived fibroblasts at the same level as in the addition of serum under serum-free conditions. [Structure] MCDB153 medium is used as a basal medium, to which calcium ion of 1.8 mM or more and magnesium ion of 0.8 to 1.2 mM are added to obtain a serum-free culture medium. Using this medium, primary culture and subculture of animal-derived fibroblasts can be performed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a good serum-free culture medium for animal-derived fibroblasts and a culture method, which is an optimal culture system for accurately analyzing the influence of various factors on fibroblasts. Is provided.

[0002]

2. Description of the Related Art Conventionally, animal-derived fibroblasts have been cultured by adding animal serum such as fetal calf and calf to a basal medium prepared by adding vitamins to amino acids like MEM medium of Eagle. I've been told.

In recent years, attempts have been made to culture without adding animal serum, but instead adding cell growth factors, cell adhesion factors, carrier proteins, antioxidants, protease inhibitors and the like. There is. For example, for serum-free culture of human fibroblasts, RITC80-7, MCDB series, HamF-12 / DME 1: 1 mixed medium and the like are used as basal medium, and insulin and EGF (Ep (Ep) are used as cell growth factors.
idermal Growth Factor, PDGF (Platelet-Derive)
d Growth Factor), bFGF (basic Fibroblast Grow)
th Factor), transferrin etc. as a carrier protein, and fibronectin as a cell adhesion factor have been developed.

[0004]

However, among the above techniques, in the culture method using a medium to which serum is added, since many substances are contained in the serum and their actions are complicated and diversified, Due to the large difference in serum lots, it was difficult to analyze the experimental results regarding the properties and physiology of fibroblasts, the influence of drugs and the like.

Further, even in a culture method under serum-free conditions, it is difficult to obtain the same level of growth as when adding serum, and it is necessary to add various components to the basal medium in order to improve the growth. However, it still hinders the analysis of the action of drugs and the like.

[0006]

[Means for Solving the Problems] Therefore, we studied to establish a culture system for animal-derived fibroblasts in which the types and amounts of the components added to the basal medium were reduced as much as possible. As a result, the MCDB153 medium, which is a high amino acid medium, was used as the basal medium, and a system in which simple inorganic ions such as calcium ion of 1.8 mM or more and magnesium ion of 0.8 to 1.2 mM were added to the medium were favorable. The inventors have found that fibroblast proliferation can be obtained, and completed the present invention.

The amount of calcium ion added is
1.8 mM or more is suitable. Figure 1 shows the basal medium MCD
The amount of magnesium ion added to the B153 medium was 1.
0 mM, and the addition amount of calcium ion is 0.1 mM
The figure shows the growth of human fibroblasts when added in the range of 1.8 mM. From this figure, it is recognized that 1.8 mM of calcium ion needs to be added in order to obtain the same level of proliferation as when serum was added.

On the other hand, the amount of magnesium ion added is 0.
8 to 1.2 mM is suitable. In FIG. 2, MCDB153
The amount of calcium ion added to the medium was 1.8 mM, and the amount of magnesium ion added was 0.5 to 1.5 mM.
It shows the growth of human fibroblasts when added in the range. As is clear from this figure, sufficient growth was not obtained with the addition of less than 0.8 mM magnesium ion,
The addition of 1.2 mM or more rather inhibits the proliferation of fibroblasts and is not preferable.

The addition of EGF or insulin to the medium of the present invention can further promote cell growth.

[0010]

As described above, MCDB153 medium is used as the basal medium, and calcium ion of 1.8 mM or more and magnesium ion of 0.8 to 1.2 mM are added to the medium, and fibroblasts derived from various tissues of humans and animals are derived. When cells are cultured under serum-free conditions, as shown in FIG. 1, it is possible to obtain the same level of proliferation as when serum is added. In the case of culturing human skin fibroblasts, the number of cells rapidly increases after 2 days of culturing, and almost the same growth curve as that when serum was added can be obtained.

The medium and culture method according to the present invention are
It can be used in both primary culture and subculture of fibroblasts, and good results are obtained.

[0012]

EXAMPLES The present invention will be described in more detail with reference to Examples.

First, as an example of the present invention, Table 1 shows the composition of a serum-free culture medium for human dermal fibroblasts. The amount of calcium ions added to this medium was 1.8 mM, and the amount of magnesium ions added was 0.98 mM.

[Table 1]

Human fibroblasts were primarily cultured using the above medium. The method is as follows.

The collected human skin tissue is washed with a phosphate buffered saline solution (PBS (-)) and then finely cut in the PBS (-) solution. Then, it was washed several times with PBS (-) to make fine slices, and
1 mg / ml trypsin, 30 with 0.02% EDTA solution
After treatment for 1 minute, the solution after filtration is centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet, and centrifugation is repeated 2-3 times to remove trypsin. The cell pellet is then suspended in the culture medium.

On the other hand, 1% of fibronectin with PBS (-)
Dilute to 0 μg / ml and overlay on a petri dish. The mixture is allowed to stand at 37 ° C. for 30 minutes, the fibronectin solution is removed by suction, and washed twice with PBS (−). After adding the medium according to the present invention, adding about 1 million of the above isolated cells,
After culturing for 2 to 3 hours in a carbon dioxide incubator, the non-adherent cells and the residue of the tissue are removed by suction. Subsequently, a new medium was added, the medium was transferred to an oxygen-carbon dioxide incubator, and the culture was further continued.

At this time, the same culture was carried out using DMEM medium supplemented with 10% calf serum as the culture medium, and Comparative Example 1 was used. MCDB153 medium was supplemented with 0.1 mM calcium ion and 0.1 mM magnesium ion. The case of culturing in the same manner as that of Comparative Example 2 was used.

The manner of proliferation of fibroblasts in this example is shown in FIG. After the start of culture, the number of adhesive fibroblasts was 2
Although the number was about 10,000, it increased to about 30,000 after 2 days, then rapidly increased to 120,000 after 6 days, and the same level of proliferation as in Comparative Example 1 to which serum was added was observed. . On the other hand, in Comparative Example 2 in which the concentration of calcium ions and magnesium ions was low, almost no proliferation of fibroblasts was observed even after culturing for 8 days.

[0019]

As described above, according to the present invention, it is possible to reduce the types and amounts of the added components under serum-free conditions as much as possible.
It has become possible to specifically and efficiently culture animal-derived fibroblasts.

[Brief description of drawings]

FIG. 1 is a diagram showing the effect of calcium ions in serum-free culture of fibroblasts.

FIG. 2 is a diagram showing the influence of magnesium ions in serum-free culture of fibroblasts.

FIG. 3 is a diagram showing a difference in proliferation between the example of the present invention and Comparative examples 1 and 2.

[Explanation of symbols]

1 When 10% calf serum was added to DMEM medium 2 When MCDB153 medium was added with 1.8 mM calcium ion and 1.0 mM magnesium ion 3 MCDB153 medium was added with 1.0 mM calcium ion and 1.0 mM magnesium ion When added 4 When added 0.1 mM calcium ion and 1.0 mM magnesium ion to MCDB153 medium 5 Example 1 6 Comparative example 1 7 Comparative example 2

Claims (2)

[Claims] 1. A medium for serum-free culture of animal-derived fibroblasts, which comprises MCDB153 medium as a basal medium and calcium ions of 1.8 mM or more and magnesium ions of 0.8 to 1.2 mM are added. 2. A MCDB153 medium containing calcium ions in an amount of 1.8 mM or more and magnesium ions in an amount of 0.8 to 0.8.
1.2 mM, and a serum-free culture method of animal-derived fibroblasts, which comprises culturing under serum-free conditions.