JP2003093051A - Method for cell culture and substrate for cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for cell culture having improved rate of proliferation using a highly safety and easily purchasable material, and to provide a substrate for cell culture used in the method. SOLUTION: The method uses a collagen extracted by acetic acid from the foot part of chicken as the substrate for the cell culture. The collagen used as the substrate for the cell culture can be used as a solution dissolved in acetic acid, and a coated film obtained by covering with the solution and subjecting to a UV irradiation is preferably used as the substrate of the cell culture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture method and a cell culture substrate composed of collagen extracted with acetic acid from chicken foot used in the method.

[0002]

2. Description of the Related Art In recent years, due to remarkable progress in culture technology, it has become possible to culture cells of animal tissues, organs, etc. in vitro. Cell culture is used for metabolism of various organs, hormone response, etc. Biochemical research, basic research on cell growth suppression, differentiation, morphology, aging, cancer, etc., search for physiologically active substances produced by cells (eg hormone-like substances, growth factors, etc.), screening system for new drug development , Drug safety test,
Widely used in fields such as clinical examinations. The growth mode of animal cells is roughly classified into adhesion-independent cells and adhesion-dependent cells. Many animal cells are the latter adhesion-dependent cells and cannot survive in the absence of a scaffolding culture substrate. Glass culture dishes and plastic culture dishes that have undergone special treatment are widely used as culture substrates that serve as scaffolds for cells, but in this culture system, cell proliferation, induction of differentiation, morphogenesis, long-term culture, etc. It has been reported that there are many cases in which the cells are unable to retain the intrinsic functions of the cells.

Therefore, a culture method utilizing a collagen matrix that mimics the cellular environment in the living body has been considered. This culturing method is a method of culturing cells on a collagen film, and it has been confirmed that many cells are effective in terms of proliferation and function retention. The collagen used to prepare the collagen film is type I collagen solubilized with acid or enzyme from connective tissue of animals or the like. A collagen film is obtained by coating this collagen on a culture dish and drying. Collagen, which is solubilized from bovine or porcine connective tissue and extracted, is used as collagen serving as such a cell culture substrate.
In recent years, its use has become difficult due to problems such as BSE (bovine spongiform encephalopathy) and foot-and-mouth disease. In addition, there is a demand for a cell culture method with further improved cell growth efficiency as compared with the cell culture methods using these conventional cell culture substrates.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves these problems, uses a raw material that is easily available and extremely safe, and is used for a cell culture method with improved cell growth efficiency, as well as for the method. The purpose is to provide a cell culture substrate.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the above problems can be solved by using collagen extracted with acetic acid from the foot portion of chicken as a cell culture substrate. Is completed. That is, the present invention has the following configurations. 1. A method for cell culture, which comprises using collagen extracted from chicken foot portions with acetic acid as a substrate for cell culture. 2. 2. The cell culture method according to 1, wherein the collagen is mainly composed of type I collagen. 3. 3. The cell culture method according to 1 or 2, wherein a solution of collagen dissolved in acetic acid is used as a cell culture substrate. 4. 4. The cell culture method according to any one of 1 to 3, wherein a coating film formed by coating a solution of collagen dissolved in acetic acid and irradiated with ultraviolet rays is used as a cell culture substrate. 5. A cell culture substrate composed of collagen extracted from the foot portion of a chicken with acetic acid. 6. 6. The cell culture substrate according to 5, wherein the collagen is mainly composed of type I collagen. 7. 7. The cell culture substrate according to 5 or 6, which is used as a solution of collagen dissolved in acetic acid.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as a substrate for cell culture, use is made of collagen having type I collagen as a main component extracted with acetic acid from chicken tendon (hereinafter referred to as "chicken-derived collagen"). Is characterized by.
Cows and pigs take several years to grow, but in the case of chickens, they grow in 42 days, so fresh collagen can be easily obtained at all times, and there is no risk of BSE or foot-and-mouth disease, which is extremely safe. is there. Moreover, the body temperature of chickens is higher by about 2 to 4 ° C. than that of cattle and pigs, and the obtained collagen is more stable to heat than that derived from cattle or pigs. In addition, cell culture efficiency is improved when used as a cell culture substrate.

The chicken-derived collagen can be stored as a lyophilized product. To use it as a cell culture substrate, for example, an aqueous solution prepared by dissolving the lyophilized collagen with acetic acid is used in a culture dish (for example, a 96-well plate). ), Etc., and thinly stretched, and then dried at a relatively low temperature to prevent denaturation of collagen to form a collagen film on a culture dish and used for cell culture.
At that time, the pH of the aqueous solution is about 1 to 5, preferably 1.
Adjust to about 5 to 3.0, collagen concentration is 1 to 10
It is preferably about mg / mL.

By irradiating the collagen film thus formed with ultraviolet rays, the cell growth efficiency can be further improved. The wavelength of the ultraviolet rays to be applied is not particularly limited, and for example, the wavelength of 190 to 400 nm is applied. UV irradiation time is 30 minutes to 2
It is preferably about 4 hours.

[0009]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the following specific examples do not limit the present invention. (Preparation of cell culture dish) 0.3 g of freeze-dried collagen derived from chicken was dissolved in 80 ml of ultrapure water, and then pH was adjusted with acetic acid.
While adjusting to 3, 20 ml of ultrapure water was added to prepare an aqueous solution having a collagen concentration of 3.0 mg / ml (hereinafter referred to as "chicken acetic acid solution"), and this solution was added to 0.20 μm.
m filter. Solution preparation and filtration is 10
It was carried out at a temperature of ℃ or less. In addition, except that hydrochloric acid was used instead of acetic acid, collagen concentration was 3.0 mg / m in the same manner.
An aqueous solution of 1 (hereinafter, referred to as "chicken hydrochloric acid solution") was prepared and similarly filtered.

Each of the obtained collagen solutions was poured into a 96-well culture dish, spread thinly, and then dried at a low temperature to prevent denaturation of collagen to prepare a culture dish coated with a collagen film. 9 for comparison
A 6-well culture dish was coated with a commercially available 0.3% aqueous solution (pH 3) of bovine collagen and porcine collagen for cell culture, and a culture dish without collagen coating (0.3% Coated with bovine albumin solution: hereinafter referred to as "uncoated culture dish") was prepared. These culture dishes were prepared by irradiating ultraviolet rays having a wavelength of 254 nm for 4 hours to cross-link the coating and non-irradiated ones.

(Example 1: Culture test of mouse colon cancer cells) A chicken acetate solution-coated culture dish (96) which has not been irradiated with ultraviolet rays.
Mouse colon cancer cells Colon26 in 0.
5 × 10 ³ cells / well were seeded. Then, RPMI
The cell proliferation of the culture dish was examined by culturing 1640 medium in a CO ₂ incubator at a temperature of 37 ° C. for 1 to 3 days and counting the number of cells. In addition, cell proliferation was examined in the same manner for chicken hydrochloric acid solution-coated culture dishes, bovine collagen-coated culture dishes, and uncoated culture dishes that had not been irradiated with ultraviolet light. The results are shown in FIG.

Next, FIG. 2 shows the results of similarly examining the cell growth properties by using ultraviolet irradiation culture dishes instead of the ultraviolet non-irradiation culture dishes, respectively. 1 and 2, the horizontal axis represents the number of culture days and the vertical axis represents the cell number. In addition, A represents a chicken acetate solution-coated culture dish, B represents a chicken hydrochloric acid solution-coated culture dish, C represents bovine collagen-coated culture dish, and D represents an uncoated culture dish. According to FIG. 1 and FIG. 2, the chicken acetic acid solution-coated culture dishes, both unirradiated and irradiated with ultraviolet rays, have excellent cell growth properties as compared with bovine collagen-coated culture dishes. In addition, the chicken hydrochloric acid solution-coated culture dish has the same cell proliferation property as that of the bovine collagen-coated culture dish when it is not irradiated with ultraviolet rays.
Those irradiated with ultraviolet rays have better cell growth properties than bovine collagen-coated culture dishes.

(Example 2: Culture test of human normal umbilical vein endothelial cells) In the same manner as in Example 1, various culture dishes (96
Well) with 0.5 × 10 ⁵ normal human umbilical vein endothelial cells
Seed individually / well and CO ₂ using CS-C medium
Incubate at 37 ° C for 1 to 4 days in an incubator,
The cell proliferation of the culture dish was examined by counting the number of cells. The results of culturing using a culture dish that has not been irradiated with ultraviolet rays are shown in FIG. 3, and the results of culturing using a culture dish that has been irradiated with ultraviolet rays are shown in FIG. 3 and 4, A is a chicken acetate solution-coated culture dish, B is a chicken hydrochloric acid solution-coated culture dish, C is bovine collagen-coated culture dish, D is an uncoated culture dish, E is a porcine collagen-coated culture dish, and F is Represents the culture results for a culture dish coated with a commercially available adhesion factor. According to these results, the chicken acetic acid solution-coated culture dish, both unirradiated and irradiated with ultraviolet light, has a superior cell growth effect as compared with other culture dishes.

(Example 3: Culture test of human normal skin fibroblasts) In the same manner as in Example 1, 0.5 × human normal skin fibroblasts were placed in various culture dishes (96 wells) irradiated with ultraviolet rays. 10 ⁵ cells / well were seeded and 1 to 37 at a temperature of 37 ° C. in a CO ₂ incubator using CS-C medium.
After culturing for 3 days, the cell proliferation of the culture dish was examined by counting the number of cells. Results are shown in FIG. In FIG. 5, A represents the culture results of the chicken acetic acid solution-coated culture dish, B represents the chicken hydrochloric acid solution-coated culture dish, C represents the bovine collagen-coated culture dish, and F represents the adhesion factor-coated culture dish. According to this result, the chicken collagen-coated culture dish has an excellent cell growth effect as compared with other culture dishes.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, a cell culture method having an improved cell growth effect is provided by using collagen extracted with acetic acid from chicken foot as a cell culture substrate, which is easily available and extremely safe. To be done. In particular, by using a solution of chicken collagen dissolved in acetic acid as a cell culture substrate, and further by using as a cell culture substrate a film constituted by the chicken collagen irradiated with ultraviolet rays, the cell growth effect is further improved. A method for culturing cells is provided.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of culturing mouse colon cancer cells using a UV non-irradiation culture dish.

FIG. 2 is a diagram showing the results of culturing mouse colon cancer cells using an ultraviolet irradiation culture dish.

FIG. 3 is a view showing the results of culturing human normal umbilical vein endothelial cells using a UV non-irradiation culture dish.

FIG. 4 is a diagram showing the results of culturing mouse colon cancer cells using an ultraviolet irradiation culture dish.

FIG. 5 is a view showing the results of culturing human normal skin fibroblasts using an ultraviolet irradiation culture dish.

Continued front page    (72) Inventor Kazuo Maruyama             5-15-12 Aihara, Sagamihara City, Kanagawa Prefecture F-term (reference) 4B065 AA91X AA93X BC41 CA44

Claims (7)

[Claims] 1. A method for cell culture, which comprises using collagen extracted from the foot portion of a chicken with acetic acid as a substrate for cell culture. 2. The cell culture method according to claim 1, wherein the collagen is mainly composed of type I collagen. 3. A solution in which collagen is dissolved in acetic acid is used as a cell culture substrate.
The cell culture method according to. 4. A cell culture substrate, which is obtained by irradiating a coating film formed by coating a solution of collagen with acetic acid with ultraviolet rays, as a cell culture substrate.
4. The cell culture method according to any one of 3 above. 5. A cell culture substrate composed of collagen extracted from the foot portion of a chicken with acetic acid. 6. The cell culture substrate according to claim 5, wherein the collagen is mainly composed of type I collagen. 7. The cell culture substrate according to claim 5 or 6, which is used as a solution in which collagen is dissolved in acetic acid.