JPH08168372A - Collagen coated cell culturing apparatus and method for producing same - Google Patents

Abstract

PURPOSE: To provide the culturing apparatus capable of being preserved for a long period in a room temperature by reacting a specific primary aminosilane coupling agent with the surface of a substrate containing hydroxyl groups, neutralizing with an acidic collagen solution to form the collagen coating by binding the amino groups to the collagen. CONSTITUTION: This collagen coated cell culturing apparatus comprises reacting a primary aminosilane coupling agent of formula I X, Y are each a hydrolyzable group such as an alkoxy, chloro, acetoxy, acetylamino or propenoxy; (n) is 2 or 3; R is formula II or III [(x), (y) are each 0-20; (m) is 0 10]} with the surface of a substrate having hydroxyl groups or the hydroxy groups introduced thereto by an oxidizing treatment with a physicochemical means to bind the aminosilane to the surface of the substrate, then neutralizing the aminosilane while contacting the acidic solution of a collagen, and coating the collagen to the surface by binding the amino groups of the aminosilane with the collagen to obtain the object culturing apparatus.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention mainly relates to the field of cell culture, and relates to a cell incubator having a culture surface coated with collagen in order to enhance cell culturing property, and a method for producing the same.

[0002]

2. Description of the Related Art Cell culture equipment such as petri dishes, flasks, multi-plates, beads, balls, or coverslips (sheet-shaped culture pieces) for use in culture vessels. Culture carriers (hereinafter, collectively referred to as cell incubators) are commercially available. In these cell incubators, a polystyrene molded article is mainly subjected to low temperature plasma treatment, corona discharge treatment or the like to impart hydrophilicity thereto.

These anchorage cells are widely used for culturing anchorage-dependent cells such as fibroblasts, smooth muscle cells, vascular endothelial cells and corneal cells, regardless of cell line or primary cell. . In addition, as blood cells, established lymphocytes NS-1, MOLT-4, HUT78, MT-
It is widely used for so-called anchorage-dependent floating cells such as No. 4 and the like. However, depending on the type of cell, although cell growth is observed on these cell incubators, the cell growth is insufficient or the cell growth form and function are poor. This is particularly noticeable in primary culture.

Such problems can be dealt with by coating the culture surface with an extracellular matrix such as collagen, gelatin, fibronectin, laminin, vitronectin, and elastin to enhance the adhesion and proliferation of cells. Many. Among them, collagen is generally widely used. Generally, as a method for coating collagen, a collagen solution is purchased as a reagent, aseptically diluted to an appropriate concentration with a phosphate buffer solution, etc., and the culture solution is covered with the diluted solution on a sterile Petri dish as described above. The method is that a predetermined amount is dispensed, left still for 1 to 2 hours, and sometimes 24 hours, and after removing the solution, it is washed with sterile pure water and used immediately.

In this way, even if the collagen coat for imparting the culturing property is applied, the effect of the coat changes with time, and eventually the effect is lost. When sterilizing a collagen-coated cell incubator, radiation sterilization such as γ-ray sterilization is generally performed, but this γ-ray sterilization also reduces the effect of collagen. Further, the cell culture device coated with collagen is used immediately after coating or stored in a refrigerated form, but the storage period is about 6 months at the longest.

Most of the conventional cell incubators are made of polystyrene, and collagen is physically adsorbed even by the above coating method, and the cells can be cultured without any trouble. It was However, recently, due to the diversification of culture morphology, the base material to be coated with collagen is also diversified, and collagen cannot be adsorbed depending on the material of the base material. Therefore, conventional coating methods can be used for cell culture. There are many cases in which it is not possible.

[0007] For example, silicone rubber has features such as low cytotoxicity, good transparency, and good gas permeability, and is widely applicable as a culture substrate in the field of cell culture. As one example thereof, the present inventors have made an invention of a cell incubator characterized in that the bottom surface inside the container body is coated with silicone rubber, and the silicone rubber portion can be peeled off, and JP-A-6-181740. Disclosed in Japanese Patent Publication No. However, if you try to coat the silicone rubber with collagen, the conventional method described above
It is difficult to coat the surface of silicone rubber with collagen. As another coating method, there is a method in which a fixed amount of collagen solution is dispensed into an incubator and dried to coat collagen, but with this method, it is difficult to keep the coating layer uniform, and , The collagen coating layer peels off during the culture, which makes it difficult to use for long-term culture.

[0008]

DISCLOSURE OF THE INVENTION The present invention is intended to solve such problems in a collagen-coated cell incubator and a collagen-coated cell, and its object is to provide a base material such as silicone rubber. Another object of the present invention is to provide a collagen-coated cell culture device capable of exerting the effect of collagen even above and having good long-term storage stability.

[0009]

As a result of intensive studies to achieve the above object, the present inventors have introduced an amino group on the surface of a base material, thereby eliminating the need for using a crosslinking agent. Also cross-links with amino groups on the surface of the base material and cross-links between collagens, so stable coating of collagen on the surface of the base material is possible even when the amount of amino groups introduced on the base material surface is small. The present invention has been completed and the present invention has been completed.

That is, the present invention is a collagen-coated cell incubator characterized in that a collagen layer is formed on the surface of a base material having an amino group, and the amino group and the collagen are chemically bonded. In the case of a base material having no hydroxyl group, an amino group is introduced by bonding a primary aminosilane to the surface of the base material having a hydroxyl group.

The second invention is that it has a hydroxyl group,
Or the substrate surface by introducing a hydroxyl group by the oxidation treatment by physicochemical means, the general formula _{NH 2 -R-Si-X n} Y 3-n ( wherein, X and Y are alkoxy groups, or a chloro group, acetoxy group , an acetylamino group, a hydrolyzable group such as propenoxy group, n represents 2 or 3, R is - [(CH _{2) x} -NH] _m - (CH _{2) y} - or -CONH - [(CH _{2) x} -NH] _m - (CH _{2) y}
Where x and y are each 0 or an integer of 20 or less, and m is 0.
Or an integer of 10 or less. ) Is reacted with a primary aminosilane coupling agent to bind aminosilane to the surface of the base material, and then neutralized while contacting with an acidic collagen solution to bind the amino group of aminosilane to collagen. And a collagen coating method.

The present invention will be described in more detail below. The shape of the target culture container is not particularly limited, but a Petri dish, a plate having a plurality of wells, or the like is used. Further, it may be applied to a culture container or the like provided with a coating on the bottom surface inside the container body, which can be easily peeled off and becomes a small piece for culture. The type of base material is not particularly limited,
Considering handling, etc., it is preferable that it is plastic. Further, since it is necessary to observe with a microscope in the process of cell culture, it is necessary that it is transparent, and as such a resin, polystyrene, polyester,
Examples include polypropylene, polyethylene, polyvinyl chloride and the like. Regarding the incubator provided with the above-mentioned covering portion, the material of the covering portion has elasticity and flexibility like rubber, has transparency, has no cytotoxicity, and has water resistance. From the above, silicone rubber is preferable.

Next, with respect to the collagen coating procedure,
Silicone rubber is described as an example. In the case of silicone rubber, since adsorption of proteins is poor, it is difficult to coat by adsorption, which is generally used.

First, an amino group that binds to collagen is introduced on the surface. As the procedure, first, a physicochemical oxidation treatment is performed to introduce a hydroxyl group on the surface. As the method of this oxidation treatment, ultraviolet irradiation, electron beam irradiation, γ-ray irradiation,
Examples include corona discharge treatment, low frequency or high frequency low temperature plasma treatment. Among these, a low-frequency or high-frequency low-temperature plasma discharge treatment, which is simple and not limited in shape, has no problem in discarding the oxidation reaction reagent, and is easy to control the introduction density of hydroxyl groups, is desirable.

The primary aminosilane coupling agent used in the present invention has a general formula of NH ₂ —R—Si—X _n Y _3-n (wherein X and Y are alkoxy groups, chloro groups, acetoxy groups, acetyl groups). amino group, a hydrolyzable group such as propenoxy group, n represents 2 or 3, R is - _[(CH 2) _x -NH] _m - (CH _{2) y} - or -CONH - [(CH _{2) x} -NH ] _M- (CH ₂ ) _y
Where x and y are each 0 or an integer of 20 or less, and m is 0
Or an integer of 10 or less. ), For example, γ-aminopropyltrimethoxysilane, N-β (aminomethyl) γ-aminopropyltrimethoxysilane, γ- (diethylenetriamino) propyltrimethoxysilane, γ
-Ureidopropyltrimethoxysilane and the like can be mentioned as suitable examples, but instead of trimethoxysilane, which is a hydrolyzable group, triethoxysilane, methyldimethoxysilane, ethyldiethoxysilane, trichloro group, triacetoxy group. It is also effective for derivatives having

As a method for introducing the primary aminosilane to the surface of the base material by the condensation reaction between the hydroxyl group on the surface of the base material and the primary aminosilane coupling agent, for example, the above-mentioned aminosilane coupling agent is added to methanol, ethanol, acetone, Dilute to a ratio of 0.1 to 98% by weight in water or the like alone or in a mixed solvent for 5 minutes to 72 hours, preferably in a solvent having a water: methanol ratio of 0 to 80: 100 to 20. After diluting to a ratio of -50% by weight, immersing or pouring for 1 to 48 hours for reaction, and then washing with distilled water or a buffer solution, the reaction temperature is 4 to 65 ° C, preferably 24 to 60 ° C is good. Under such conditions, amino groups are introduced into the surface of the base material at a density of 0.1 to 20 mmol / cm ² .

Next, as a method for coating collagen on the surface of the base material into which amino groups have been introduced as described above, an acidic solution of acid-solubilized collagen is used in water, ethanol, methanol or the like alone or in a mixed solvent. , Diluted to a collagen concentration of 0.001-0.3% by weight,
Preferably, when diluting the acidic solution of acid-solubilized collagen, the ratio of ethanol: water is 0 to 75: 100 to 25.
It is diluted to 0.01-0.1% by weight in a solvent in the range of 0.1 to 0.1% by weight, and after dipping or pouring, an aqueous solution of sodium hydroxide or sodium carbonate is poured to make it neutral or weakly alkaline. . At this time, the collagen molecules chemically react with each other, and the amino group introduced into the base material and the collagen molecule cause a chemical reaction, so that the base material and the collagen are firmly bonded. The collagen coat layer that is tightly bound and coated in this way is extremely stable and has excellent storage stability, stability in radiation sterilization, and stability in long-term culture when actually used in cell culture. ing.

[0018]

EXAMPLES Next, the present invention will be described more specifically by way of examples. [Example 1] A culture surface of a polystyrene tissue culture dish having a diameter of 35 mm (MS-10350 manufactured by Sumitomo Bakelite Co., Ltd.) was treated with a primary aminosilane coupling agent, introduced with an amino group, washed, and then dried at 45 ° C. Collagen acidic solution adjusted to 0.015% collagen concentration was dispensed, neutralized by adding a mixed alkaline solution of sodium hydroxide and sodium carbonate, left for 1 hour, and then washed with pure water. Then, the pure water was removed by suction, dried in a dryer at 37 ° C., and subjected to a culture test.

[Example 2] A silicone rubber layer was formed on the culture surface of the same polystyrene tissue culture dish as in Example 1, and oxygen plasma treatment was applied to the surface to introduce hydroxyl groups. This was treated with a primary aminosilane coupling agent in the same manner as in Example 1 to introduce an amino group,
The cells were coated with collagen and subjected to a culture test.

Comparative Example 1 The same polystyrene tissue culture dish as in Example 1 had a collagen concentration of 0.03%.
The acidic collagen solution of 1 was dispensed, left to stand at room temperature for 12 hours, washed with pure water, the pure water was removed by suction, and dried in a dryer at 37 ° C. to be used for a culture test.

Comparative Example 2 A polystyrene dish having a diameter of 35 mm was subjected to corona discharge treatment to make the culture surface hydrophilic, sterilized with ultraviolet rays, and immediately subjected to a culture test.

Each sample was stored or sterilized under the following conditions to evaluate the culturing property. Storage test under elevated temperature Humidity 60% in constant temperature bath at 50 ℃, 60 ℃ and 70 ℃
The cell culturing property was comparatively evaluated for the samples that were kept at 4 ° C. for 16 hours and stored at 4 ° C. High Humidity Storage Test The cell culturing properties were compared and evaluated between those stored at 37 ° C. and 100% humidity for 1 month and those not stored. Storage test at room temperature 6 months in a room without air conditioning ('94 .3-'94.9)
The cell culture properties of the preserved and non-preserved cells were compared and evaluated. γ-ray Sterilization Test After sterilizing by irradiating with γ-ray at doses of 0 kGy, 10 kGy and 25 kGy, cell culturing property was evaluated immediately.

The method for evaluating the cell culturing property of each sample was as follows. Evaluation of Collagen Coat with Hep G2 Cells With respect to each petri dish of Examples 1 and 2, Comparative Example 1 and Comparative Example 2, Hep G2 cells (human hepatoma-derived cell line) were used to examine cell growth morphology (extension degree). It was As the medium, 500 ml of Dulbecco's modified MEM medium added with 50 ml of fetal bovine serum was used, and 2 ml each was seeded at a concentration of 5 × 10 ⁴ cells / ml on each petri dish to invert the growth form in 2 days of culture. It was observed with a microscope and compared with the petri dish of Comparative Example 2. The evaluation results were as shown in Table 1.

The evaluation results are all shown by the symbols compared with each other using Comparative Example 2 as a standard (±). ++: Very effective, +: Effective, ±: Same as Comparative Example 2, − : Inferior to Comparative Example 2

[0025]

[Table 1]

Evaluation of Collagen-Coated V79 Cells For the petri dishes of Examples 1 and 2 and Comparative Examples 1 and 2, V
The cell growth morphology (extension degree) was examined using 79 cells (fibroblast-derived cell line). As the medium, 500 ml of Dulbecco's modified MEM medium added with 50 ml of fetal bovine serum was used, and 2 ml each was seeded at a concentration of 1 × 10 ⁴ cells / ml to each petri dish to invert the growth form in 3 days of culture. It was observed with a microscope and compared with the petri dish of Comparative Example 2. The evaluation results were as shown in Table 1. The evaluation result is
All were compared using Comparative Example 2 as a reference 100.

[0027]

[Table 2]

As can be seen from the results of the measurements and observations shown in Tables 1 and 2, the collagen-coated incubator of the present invention, even when exposed to high temperature and high humidity during storage,
Moreover, it is clear that the effect of the collagen coat is maintained without being deactivated even when exposed to γ-rays.

[0029]

EFFECTS OF THE INVENTION According to the present invention, the collagen-coated incubator can be exposed to high temperature and high humidity during storage.
In addition, the effect of the collagen coat can be maintained without being deactivated even when exposed to γ-rays, and it requires refrigeration for transportation, but can be stored and handled at room temperature. It is suitable as a collagen-coated cell incubator and a method for producing the same, because the control of sterility in the above can be simplified.

Claims (6)

[Claims] 1. A collagen-coated cell incubator, which comprises forming a collagen layer on the surface of a base material having an amino group and chemically binding the amino group and collagen. 2. The collagen-coated cell culture device according to claim 1, wherein the amino group is introduced by binding a primary aminosilane to the surface of a base material having a hydroxyl group. 3. The collagen-coated cell culture device according to claim 1, wherein the base material is silicone rubber. 4. The silicone rubber as a base material is coated on the bottom surface in the container body in a thickness of 0.05 to 2 mm so that it can be peeled off. Collagen coated cell incubator. 5. The surface of a substrate having a hydroxyl group or having a hydroxyl group introduced by an oxidative treatment by physicochemical means has the general formula NH ₂ —R—Si—X _n Y _3-n (wherein X and Y alkoxy group, or a chloro group, acetoxy group, an acetylamino group, a hydrolyzable group such as propenoxy group, n represents 2 or 3, R - [(CH _{2) x} -NH] _m - (CH _{2) y} - or -CONH - [(CH _{2) x} -NH] _m - (CH _{2) y}
Where x and y are each 0 or an integer of 20 or less, and m is 0
Or an integer of 10 or less. ) Is reacted with a primary aminosilane coupling agent to bond aminosilane to the surface of the base material, and then neutralized while contacting with an acidic collagen solution to bond the amino group of aminosilane to collagen. And a method of producing a collagen-coated cell incubator, which comprises coating collagen. 6. The collagen coat according to claim 5, wherein the oxidation treatment by physicochemical means is any one of low temperature oxygen plasma, low temperature air plasma, low temperature argon plasma, and corona discharge treatment. Method for manufacturing cell culture device.