JPH11127843A - Colored container for culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container for culture capable of simply emitting light and usable for measuring fluorescence without deteriorating cell culturing properties. SOLUTION: This colored container for culture is obtained by molding thereof from a light untransmitting resin and further coating the culturing surface with a glycoprotein such as a cell adhesion factor collagen, gelatin, a fibronectin or a laminin or a synthetic peptide such as poly-L-lysine in order to enhance the cultured cell adhesion.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture vessel used for tissue culture or cell culture using a luminescent or fluorescent substance as a labeling substance.

[0002]

2. Description of the Related Art Generally, in the field of tissue culture and cell culture, a culture vessel is made of a material such as glass or plastic having high optical transparency and low distortion so that the state of the tissue and cells can be conveniently observed. An assembly of containers in which a plurality of containers are integrated or combined is convenient and widely used for simultaneously operating a plurality of cell cultures. (Hereinafter, an assembly of containers is referred to as a “multiplate” and each container of the multiplate is referred to as a “well”.)

Although such a culture container is useful for observing the culture state of a tissue or a cell, there are some problems in performing measurement and analysis using the cultured cell. For example, when performing general immunoassay directly in a state where cells are present in a multiplate, a solution reaction is usually performed and the light transmittance of the solution is measured as an index. Because it is not completely optically transparent, it affects its light transmission and cannot be a correct measurement. In addition, at present, in the case of luminescence and fluorescence systems that show signs of fashion in such cell-based assays, light leaks from adjacent wells due to the transparency of the container, which makes it impossible to obtain correct measurement values. was difficult.

As described in Japanese Patent Application Laid-Open No. 58-189424, a multiplate used for luminescence and fluorescence analysis can be made of a multi-plate with a colored resin to eliminate the influence of luminescence and fluorescence of the adjacent wells described above. is what happened. In an experimental system for cell culture, a method has been used in which cells are cultured in a transparent multiplate, and then the contents including the cells are transferred to a multiplate molded with a colored resin for measurement. Japanese Patent Application Laid-Open No. 7-3410 discloses a method for solving the inconvenience of changing containers between the culture system and the measurement system.
Japanese Patent No. 64 invents a colored multiplate in which only the bottom part is transparent, invents a multiplate that can be used for light emission and fluorescence measurement by mounting a light shielding material on the bottom to prevent lateral leakage of light by observing with a microscope. I have.

However, in such a measurement system, the number of objects to be measured is increasing at present, and it is usual to use a plurality of multiplates, and in some cases, ten or more multiplates. In this case, it is not necessary to observe all the culture conditions of the cells individually, and it is general to carry out measurement by assuming that only one sheet is observed under a microscope and that the remaining cells are all in the same state. In such a case, the multiplate used for culturing and measurement is a multiplate colored over the entire surface. However, even if the resin used for molding is the same between the colored multiplate and the transparent multiplate, the colored multiplate contains a certain percentage of a coloring agent (generally titanium or carbon), and such a coloring agent is exposed on the cell culture surface. In some cases, cell cultureability may be affected.

[0006] Furthermore, the colorant is not uniformly distributed in the multi-plate due to the flow of the resin at the time of molding, whereby the cultivation of cells in one multi-plate varies. Furthermore, multi-well plates molded using plastic resin are usually used to improve cell cultureability.
The surface of the incubator is subjected to a surface treatment such as a plasma treatment, a corona treatment, and a radiation irradiation treatment. However, many colorants do not show an improvement in cell adhesion even by such treatment. From the above, it is shown that in the multiplate, it is completely impossible to represent the cell culture property of the colored plate by the multiplate having a transparent bottom surface.

[0007]

SUMMARY OF THE INVENTION The present invention provides a culture vessel capable of directly and stably performing measurement and analysis utilizing luminescence or fluorescence phenomenon in a culture vessel in culturing tissues or cells. Therefore, as a result of various studies, the present invention has been achieved.

[0008]

Means for Solving the Problems In a cell culture, a container is molded with a light-impermeable resin in order to enable measurement of a labeling substance using luminescence or fluorescence phenomenon, and further, the adhesion of the cultured cells is enhanced. A cell adhesion factor coated on the culture surface, and the cell adhesion factor to be further applied is a culture protein comprising a glycoprotein such as collagen, gelatin, fibronectin and laminin, and a synthetic peptide such as poly-L-lysine. It is a colored container.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The shape of a target culture vessel is not particularly limited, but generally, as shown in FIG. 1, a multiplate body (1) comprising a plurality of wells (13).
1) A culture vessel comprising a lid (12) is used. The members constituting the multiplate main body are required to have no cytotoxicity, water resistance, and the like. The material is not particularly limited as long as this condition is satisfied. Generally, glass or a plastic resin which is easy to process is used. However, in the present invention, the use of a plastic resin is optimal in view of easy coloring and good processability.

When an adhesive, a paint or the like is used for a member constituting the multi-plate main body, it is necessary that the multi-plate main body be cured at room temperature in addition to the above-mentioned conditions. Next, the structure of the container will be described. FIG. 2 shows a cross-sectional view of a container of one embodiment of the culture vessel using the present invention. First, the bottom and side walls are made of an opaque material to block light that may be emitted by the outside world or other wells. However, as it is, exposure of the colorant to the surface and non-uniformity due to the flow of the resin cause a problem in cell cultureability as described above. Therefore, the present applicant has applied a cell adhesion factor to the bottom surface of the well to which the cells adhere and the side surface (21) extending upward from the bottom surface, thereby forming a cell adhesion factor layer (22).
The inventors invented that cells are adhered by using a cell adhesion factor instead of the material of the plate.

The adhesive factors to be applied include various commonly used collagen, gelatin, fibronectin,
Biologically derived glycoproteins such as laminin and synthetic peptides such as poly-L-lysine are conceivable, but are not particularly limited. In addition, as a method for applying the cell adhesion factor, a method of dispensing a solution of the cell adhesion factor into a well and physically adsorbing the cell adhesion factor, a carboxyl group introduced by applying oxygen and carbon monoxide plasma treatment to the surface of the multiplate, and an ammonia Numerous surface coating methods can be applied, such as a method of chemically bonding to amino groups or the like introduced by plasma treatment. In addition, for various cell adhesion factors that cause gelation such as collagen, gelatin, laminin, etc.
It is possible to use the gel as it is, or to use it after drying the gel.

The sterilization required for the culture vessel can be performed before or after the application of the cell adhesion factor. Sterilization before application of the cell adhesion factor can be performed by either ethylene oxide gas sterilization or radiation sterilization. In the case of sterilization before application, it is necessary to apply a cell adhesion factor by filtering and sterilizing a solution of the cell adhesion factor and applying the solution in a sterile environment. The sterilization after application of the cell adhesion factor is most preferably radiation sterilization. Ethylene oxide gas sterilization is not preferable because cell adhesion factors are decomposed by ethylene oxide and high temperature.

According to the present invention described above, the cells are always stably adhered to the multiplate surface without being affected by the colorant on the surface of the multiplate, and cultured by a general cell culture method without any special operation. It is possible to do. Furthermore, in the case of analysis using a light-emitting or fluorescent system, which is the object of the present invention, since the container is made of a light-shielding material, it can be used for analysis without any special operation from the culture state.

[0014]

Next, the present invention will be described in detail with reference to examples. (Example) Polystyrene (Polystyrene M manufactured by Sumitomo Chemical Co., Ltd.)
A 96-well multiplate was injection molded from a material obtained by adding 5% of titanium oxide to −140). Irradiation of molded products 1
After sterilization with 0 kGy, commercially available bovine dermis-derived collagen type I (0.03% solution manufactured by Koken Co., Ltd. was dispensed into each well,
After being left overnight on a clean bench, the plate was washed with a sterilized phosphate buffer (pH 7.4).

Comparative Example 1 Polystyrene (manufactured by Sumitomo Chemical Co., Ltd.)
(Polystyrene M-140) 96-well multiplate was injection molded. After the molded article was sterilized by irradiation with 10 kGy of radiation, a commercially available bovine dermis-derived collagen type I (0.03% solution manufactured by Koken Co., Ltd.) was dispensed to each well, left overnight on a clean bench, and then sterilized phosphate buffer (pH 7). (Comparative Example 2) A 96-well multiplate was injection-molded with polystyrene (polystyrene M-140 manufactured by Sumitomo Chemical Co.), and the surface of the molded product was subjected to corona treatment to enhance cell adhesion. Was sterilized by irradiation with 10 kGy

Comparative Example 3 Polystyrene (Sumitomo Chemical Co., Ltd.)
A 96-well multiplate was injection molded from a material obtained by adding 5% of titanium oxide to polystyrene (M-140). The surface of the molded article was subjected to corona treatment to enhance cell adhesion. Thereafter, the molded article was sterilized by irradiation with 10 kGy of radiation. In Examples and Comparative Examples 1 to 3, human hepatoma-derived cell lines (Hep G2) were cultured, and the number of adherent cells on the first day of culture was determined as M
Measured by TT assay. The culture solution used for the culture was prepared by adding 50 ml of fetal bovine serum to 500 ml of Dulbecco's modified MEM medium.
Was seeded in each well at a concentration of 200 μl and unattached cells were washed with a phosphate buffer (pH 7.4) 24 hours after seeding, and then an MTT assay was performed. Table 1 shows the results of the ratio of the number of adherent cells to the number of cell seeds.

Table 1 Coloring Cell Adhesion Factor Cell Adhesion Rate Example Yes Yes 74.8% Comparative Example 1 No Yes 75.4% Comparative Example 2 No No 60.4% Comparative Example 3 Yes No 45.6% Regarding Comparative Example 1, it can be seen that there was almost no difference in the cell adhesion rate due to the effect of the cell adhesion factor. On the other hand, in Comparative Examples 2 and 3, the cell adhesion rate was 15
%, And it became clear that there was a difference in cultureability due to the molding resin.

[0018]

According to the present invention, cells can be stably adhered to the surface of the multiplate in the multiplate and cultured without being affected by the material, and the cells can be analyzed in the luminescence and fluorescence systems. Since the container was made of a light-shielding material, it became possible to use it directly for analysis from the culture state.

[Brief description of the drawings]

FIG. 1 is a perspective view of one embodiment of a colored container for culture according to the present invention.

FIG. 2 is a vertical sectional view of a well for culturing.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 Main body 12 Lid 13 Well 21 Colored molded product 22 Extracellular matrix coating layer

Claims (2)

[Claims] 1. In a cell culture, a container is molded with a light-impermeable resin so as to enable measurement of a labeling substance using a luminescence or fluorescence phenomenon. A colored container for culture, characterized by applying a cell adhesion factor to a culture surface. 2. The cell adhesion factor to be applied is collagen,
The culture container according to claim 1, which is a glycoprotein such as gelatin, fibronectin, laminin, or a synthetic peptide such as poly-L-lysine.