JP3406351B2 - Cell culture material and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture material such as a biocompatible material used for various medical applications and a method for producing the same, and more particularly to a cell culture material capable of improving cell adhesion and proliferation. The manufacturing method is related.

[0002]

2. Description of the Related Art Conventionally, as a cell culture material, a material for modifying the surface of a polymer material to improve the adhesion or proliferation of animal cells has been known. There is such a method. First, there is a coating treatment as a method for improving the surface of a polymeric material. In this coating treatment, the surface of the polymeric material is coated with collagen, fibronectin, etc., which is an adhesion protein, to improve the adhesiveness of cells.

As another modification method, the surface of the polymer material is treated with plasma to roughen the material,
There is a method of changing the contact angle to water to change the adhesion of cells.

Still another modification method is ion irradiation treatment. In this ion irradiation treatment, ions such as Na ⁺ , Ne ⁺ , Ar ⁺ , and N ₂ ⁺ are injected into the surface layer of the polymer material at an acceleration energy of 50 to 150 keV and an ion implantation amount of 1 × 10 ¹⁵ ions / cm ² , By generating groups, breaking the polymer main chain, and doping effects, the surface of the polymer material is modified to improve cell adhesion and proliferation (Nuclear Inst
ruments and Methodsin Phys. Res. B65, 142, 199
2).

Further, as a conventional method for controlling the sequence of cells,
The following methods are known. That is, a pattern of membrane-degrading enzyme or antibacterial substance is formed on a support, this pattern is masked with a negative pattern, and then ultraviolet rays or an electron beam is exposed from above the mask to inactivate the enzyme in the pattern site. There is a method of performing pattern culture on a support (Japanese Patent Laid-Open No. 1-291791).

[0006] In addition to this, as a method for controlling cell arrangement, there is a chemical method (artificial organs 19, 1143, 1990) in which the surface is finely modified by a photoreaction. That is, a mask was placed on a thin film of hydrophilic and hydrophobic copolymers introduced into the phenyl azide group side chain, UV irradiation was performed to remove the unexposed portion with a solvent, and then a hydrophilic polymer was placed on the hydrophobic substrate. It is a method of chemically fixing or fixing a polymer on a hydrophobic substrate to selectively grow cells only in the non-hydrophilic portion. There is also a method of forming a fine pattern on a resin substrate with an aluminum vapor deposition film and subjecting this to oxygen-reactive ion etching to perform hydrophilic treatment only on the etched portion (Polym. Reprints, Japan, 40, 671, 1991).

[0007]

In a coating treatment for modifying the surface of a polymer material, the protein is expensive and there is a problem in the adhesive strength of the protein to the substrate. Among these, when collagen is used, the adhesiveness of cells varies depending on the water content, and its control is not easy. Further, in the plasma treatment, depending on the substrate material, there is a tendency to suppress the proliferation of cells, and if the plasma treatment conditions are different, there is a problem in reproducibility of the cell culture material.

On the other hand, the ion irradiation has a problem that the apparatus is expensive and not general, and the ions can be implanted only on a limited plane. Furthermore, the method used for cell array control has a problem that usable materials are limited because ultraviolet irradiation or ion etching is performed on a specially processed surface. The present invention has been made in view of the above points, and an object of the present invention is to provide a cell culture material that is excellent in adhesiveness and proliferation and can be produced at low cost, and a method for producing the same.

[0009]

The present invention according to claim 1 has a polymer material and a carbon vapor deposition surface obtained by vapor depositing carbon on the surface of the polymer material. It is a cell culture material characterized by culturing cells in. The invention according to claim 4 is a method for producing a cell culture material, which comprises a step of preparing a polymer material and a step of vapor-depositing carbon on the surface of the polymer material to obtain a carbon vapor deposition surface.

[0010]

According to the invention described in claims 1 and 4, carbon can be vapor-deposited on the surface of the polymeric material, and the carbon can enhance the proliferation and adhesion of cells.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the drawings with reference to FIGS. First, as shown in FIG. 2A, the cell incubator 10 is of a petri dish type and is constituted by depositing carbon 12 on a polystyrene material 11.

Next, a method for manufacturing the cell incubator will be described. First, a Petri dish type cell incubator 10 made of a polymer material such as polystyrene material 11 is prepared. Next, the cell incubator 10 is placed on the bottom portion 23 of the vacuum vapor deposition device 20. In this case, the cell incubator 10 is the vacuum deposition device 2
In 0, it is arranged directly below the pair of carbon rods 21a and 21b held by the retainers 22 and 22 with a distance of about 13 cm. Each of the pair of carbon rods 21a and 21b has a diameter of 5 mm.
1a has a sharp tip, and the other carbon rod 21b has a flat tip.

As the vacuum vapor deposition apparatus 20, for example, JEE-4C type manufactured by JEOL Ltd. can be used.
With this vacuum vapor deposition device 20, a vacuum degree of 10 ⁻⁶ Torr,
A current is passed for 5 to 10 seconds under the condition of a current of 30 A to deposit carbon 12 on the surface of polystyrene material 11. In this way, as shown in FIG. 2A, the cell incubator 10 in which the carbon 12 is vapor-deposited on the surface of the polystyrene material 11 is formed.
Is obtained.

As the cell incubator 10 made of polystyrene material 11 before carbon deposition, for example, Becton Dic
A FALCON 1008 polystyrene petri dish manufactured by hinson Co., Ltd. can be used.

Further, instead of the cell incubator 10 made of the polystyrene material 11 shown in FIG. 2A, the cell incubator 10 as shown in FIG. 2B may be used for carbon vapor deposition. That is, in FIG. 2B, the petri dish type cell incubator 10 comprises a glass material 13 and a segmented polyurethane 14 coated on the surface of the glass material 13. Then, the carbon 12 is applied to the surface of the segmented polyurethane 14 by the vacuum vapor deposition device 20.
2 is vapor-deposited, and as shown in FIG. 2B, the cell incubator 10 having the carbon 12 vapor-deposited on the surface of the segmented polyurethane 14 is obtained.

In the petri dish type cell incubator 10 composed of the glass material 13 and the segmented polyurethane 14, for example, the soft segment is composed of polytetramethylene oxide and a polydimethylsiloxane compound, and the hard segment is 4,4-diphenylmethane diisocyanate. It is possible to use a segmented polyurethane manufactured by Kanegafuchi Chemical Industry. Dissolve this segmented polyurethane in tetrahydrofuran at a concentration of 5% to give an inner diameter of 2
The cell incubator 10 is obtained by applying it on a petri dish made of a glass material 13 of 6.5 mm and then drying it in a film form. This segmented polyurethane was developed as a material with good antithrombogenicity, and it has been confirmed that cells generally do not grow on this film.

Next, the carbon 12 is thus converted into the polystyrene material 11 or the segmented polyurethane material 14 in this manner.
The vascular endothelial cells were cultured using the cell incubator 10 formed by vapor deposition on the surface of the. That is, endothelial cells were detached from the bovine thoracic aorta by a method slightly modified from the method of Jaffe et al. (J. Clin. Invest., 52, 2645, 1973). For the culture, the cells were suspended in a culture medium containing 10% fetal bovine serum (RPMI-1640 manufactured by Nissui Pharmaceutical Co., Ltd.) (cell number: 5).
10,000 pieces / ml), and put these in a cell incubator 10 having carbon 12 vapor-deposited on the surface, and 2-6 in an incubator under the environment of 100% humidity, 5% CO ₂ concentration and 37 ° C. temperature.
Cultured for a day. Observation of cells is performed with a phase contrast optical microscope,
The number of cells was determined by counting the cells on a counting plate under a microscope.

As a result, it was possible to carry out culture with excellent adhesiveness and growth. In the above embodiment,
Petri dish-type incubator 10 having carbon 12 deposited on the surface
However, the cell culture material may be manufactured by depositing carbon 12 on the surface of a polymer material whose shape is not particularly determined. (Experimental Example) An experimental example of the present invention will be described below with reference to FIGS. 3 to 9. First, as shown in FIG. 3, a rectangular tin plate was placed on a part of a petri dish cell incubator 10 made of polystyrene material 11, and carbon 12 was vapor-deposited on the plate.
FIG. 3 is a diagram sketching the state of cell proliferation at this time based on microscopic observation. Of these, FIG.
It is the A section of (a). In FIG. 3, the state of cell proliferation on the third day after cell seeding is shown. As shown in FIG.
It was found that the growth of the cells 5 on the carbon-deposited surface 1 of the cell incubator 10 was superior to the growth on the carbon-undeposited surface 2 (the part where the tin plate was placed).

Next, the cells were cultured on the surface on which carbon 12 was vapor-deposited in the cell incubator 10 made of polystyrene (PS) material 11. The relationship between the number of cells and the number of culture days at this time is shown in FIG. For comparison in FIG. 4, polystyrene material 1
The relationship between the number of cells on one surface and the number of culture days is shown. As shown in FIG. 4, it was confirmed that the growth of the cells was further improved by vapor-depositing carbon 12 on the polystyrene material 11 capable of growing the endothelial cells.

Next, the segmented polyurethane (SP
U) A tin plate was placed on a part of the surface of the cell incubator 10 coated with the material 14, and carbon 12 was vapor-deposited thereon.
The state of proliferation on the third day after the cell seeding at this time is shown in FIG. Of these, FIG. 5B is an enlarged view of a portion A of FIG. As shown in FIG. 5, the cells 5 grew on the carbon-deposited surface 3 of the cell incubator 10, but the cells 5 did not grow at all on the carbon-non-deposited surface 4. This confirmed the effectiveness of carbon vapor deposition.

Next, segmented polyurethane (SPU)
The cells were cultured on the surface on which carbon 12 was deposited on the material 14. The relationship between the number of cells and the number of culture days at this time is shown in FIG. For comparison, FIG. 6 shows the relationship between the number of cells and the number of culture days on the surface of the segmented polyurethane material 14. As shown in FIG. 6, it was confirmed that the growth of cells was improved by depositing carbon 12.

Next, carbon 12 was vapor-deposited on the cell incubator 10 made of polystyrene material 11, and the detachment of the cells from the substrate surface was observed. This state is shown in FIG. In this case, the cells were cultured for 4 days on the carbon vapor deposition surface (right half 1 portion) and the carbon non-vapor deposition surface (left half 2 portion). Then, trypsin was dissolved in phosphate buffer at a concentration of 0.13%, this solution was allowed to act on the cell surface for 5 minutes, and then the cell surface was washed once with phosphate buffer. Fig. 7 (b) shows the state of the cells 5 detached from the substrate surface at this time, and Fig. 7 (a) shows the adhesion state of the cells 5 before the trypsin solution is applied.
Shown in. As shown in FIGS. 7A and 7B, carbon 1
It was confirmed that the vapor deposition of 2 increases the adhesive strength of the cells 5 against the action of trypsin.

Next, as shown in FIG. 8, a linear opening 50 is formed on the surface of the cell incubator 10 coated with the SPU material 14.
A brass mask 30 having a 0 μm groove 31 and a 50 μm groove 32 was placed (FIG. 8 (c)), carbon 12 was vapor-deposited from above, and culture was performed thereon. Figure 8 (a) shows 50μ
FIG. 8B shows the culture result when the mask 30 having the m groove 32 was used, and FIG. 8B shows the culture result when the mask having the 500 μm groove 31 was used. When the SPU material 14 is used, by selectively depositing the carbon 12 using the mask 30, two-dimensional control of cell adhesion / proliferation becomes possible. That is, in FIG.
The m-groove 32 can realize the lumen formation of the cells 5, and
In FIG. 8B, the cells 5 grow along the groove 31 by the 50 μm groove 31.

Next, as shown in FIG. 9, the inner surface of a glass tube 16 having an inner diameter of 9 mm and a length of 30 mm is coated with carbon-deposited SPU material 14, and the surface thereof is covered with cells 5 by spin culturing. Cultured for a week. In this case, the inner surface of the tube 16 could be uniformly coated with the cells 5. The rotary culture method shown in FIG. 9 can be used for producing a hybrid artificial blood vessel using vascular endothelial cells.

As described above, according to this example, since the cell growth property and the adhesiveness can be enhanced on the surface of the cell culture material, the cell culture material is used as a culture dish, artificial organ, functional biological material, and It can be effectively applied as a biomaterial.

[0026]

As described above, the first and fourth aspects are provided.
According to the invention described in (1), by vapor-depositing carbon on the surface of the polymer material, it is possible to enhance the cell proliferation and adhesion. Therefore, the cell culture material can be effectively applied as a culture dish, artificial organ, functional biological material and biomaterial.

[Brief description of drawings]

FIG. 1 is a diagram showing a vacuum vapor deposition apparatus used in a method for producing a cell culture material according to the present invention.

FIG. 2 is a diagram of the cell culture material according to the present invention used in a petri dish cell incubator.

FIG. 3 is a view showing a cell growth state when carbon is vapor-deposited on a polystyrene material.

FIG. 4 is a diagram showing the relationship between the number of cells and the number of culture days when carbon is vapor-deposited on a polystyrene material.

FIG. 5 is a view showing a cell growth state when carbon is vapor-deposited on segmented polyurethane.

FIG. 6 is a diagram showing the relationship between the number of cells and the number of culture days when carbon is vapor-deposited on segmented polyurethane.

FIG. 7 is a diagram showing how cells are detached from the substrate surface when carbon is vapor-deposited on a polystyrene material.

FIG. 8 is a view showing a cell growth state when carbon is vapor-deposited by placing a mask having an opening groove on segmented polyurethane.

FIG. 9 is a view showing a cell proliferation state when a carbon-deposited segmented polyurethane is coated on the inner surface of a glass tube.

[Explanation of symbols]

10 cell incubator 11 Polystyrene material 12 carbon 13 glass materials 14 segmented polyurethane

─────────────────────────────────────────────────── ─── Continuation of front page (56) References Tokuhyo Hira 2-501529 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12M 1/00-3/06 BIOSIS (DIALOG )

Claims (6)

(57) [Claims] 1. A cell culture material comprising a polymer material and a carbon vapor deposition surface obtained by vapor depositing carbon on the surface of the polymer material, and culturing cells on the carbon vapor deposition surface. . 2. A cell comprising a segmented polyurethane material and a carbon vapor deposition surface obtained by vapor depositing carbon on the surface of the segmented polyurethane material, wherein cells are cultured on the carbon vapor deposition surface. Culture material. 3. A cell culture material comprising a polystyrene material and a carbon vapor deposition surface obtained by vapor depositing carbon on the surface of the polystyrene material, and culturing cells on the carbon vapor deposition surface. 4. A method for producing a cell culture material, which comprises a step of preparing a polymer material and a step of vapor-depositing carbon on the surface of the polymer material to obtain a carbon vapor deposition surface. 5. A method for producing a cell culture material, which comprises a step of preparing a segmented polyurethane material and a step of vapor-depositing carbon on the surface of the segmented polyurethane material to obtain a carbon vapor deposition surface. 6. A method for producing a cell culture material, which comprises a step of preparing a polystyrene material and a step of vapor-depositing carbon on the surface of the polystyrene material to obtain a carbon vapor-deposited surface.