JP3359556B2 - Culture plate and its culture method or test method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture plate used in a bioassay method using cultured cells to determine the effect of a drug or the like and to test its toxicity.

[0002]

2. Description of the Related Art Techniques for testing and examining cells isolated from tissues have become indispensable methods in the field of life sciences, and include the diagnosis of diseases and conditions, the search for new drugs and the evaluation of drug efficacy, or It is widely used for testing environmental pollutants. Although the isolated cells may be used immediately for the test, most of them are cultured in a culture dish or a test tube by a cell culture method. Various tests are performed in this culture system.

[0003] In these assays, usually, a uniform culture system is set, and the effect is evaluated by changing the amount, concentration, etc. of the drug to be evaluated. For this reason, an incubator used for culturing is also used in a uniform and uniform manner. As this incubator, a culture dish is generally used. In order to evaluate many samples at the same time, what is called a culture plate formed by connecting the culture dishes is often used.

[0004] The cells used for evaluation are
There are adhesion-dependent cells that cannot maintain their viability unless they adhere to an incubator and cannot express normal functions, and floating cells that have little influence on the maintenance of their functions by adhesion.

[0005] In order to construct an evaluation system, these cells are cultured at a constant density in an incubator. At this time, the culture density is set under various conditions depending on the respective evaluation systems. In addition, even if the same cells are used, the conditions vary depending on the experimental system. However, in an in vitro artificial culture system, by changing the cell density, especially when a low-density culture system is used to reduce the number of cells, the function of the cells may change, or even the survival of the cells may occur. May not be maintained. This is because cell-cell interaction works in this type of culture system. This effect is thought to be due to the effects of cytokines produced by the cells, but it is also important for the adhesion-dependent cells that the cells are in contact with each other, and such a cell population creates a local culture microenvironment. Make up. In other words, reducing the number of cells makes it difficult to maintain such a culture microenvironment.

[0006]

As described above, in the conventional cell culture system, the cell density and the number of cells cannot be freely changed in order to exhibit a certain function. When the cell number and culture density are reduced, their functions are also reduced. In addition, when cells are cultured at a low density, the growth rate is also decreased, and the growth may stop and the cells may die due to apoptosis or the like.

In order to reduce the number of cells and maintain the same cell density, the culture area, that is, the shape of the culture dish must be changed. That is, when testing must be performed under multiple conditions, a large number of culture dishes or culture plates having different shapes and different culture areas must be used.
Although such a method can be applied when the number of samples is small, it cannot be said that it is entirely appropriate when a large number of samples are to be tested.

[0008]

Means for Solving the Problems A variety of methods for maintaining the same culture environment while changing the culture conditions in the same culture plate were studied. As a result, changing the culture area gave good results. The incubator obtained as a result of this study is
The culture dish in which the cell culture region is changed at an arbitrary rate is continuously formed. That is, an object of the present invention is to provide a culture plate suitable for a drug bioassay or the like, which can maintain a constant culture state while changing the number of cultured cells in a plate shape capable of simultaneously processing multiple samples. .

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The culture plate of the present invention can be of any shape as long as the culture dish is formed continuously. Commonly used ones include:
There are 6-well plates (six-well plates, hereinafter the same), 12-well, 24-well, 48-well, and 96-well plates. This one well corresponds to one culture dish, and these have approximately the same size of the entire plate and a reduced size of each well. In addition to these, those with different overall shapes are also used. Also, due to the recent trend toward miniaturization, plates (384 wells) having a smaller diameter and comprising a large number of culture dishes have begun to be used.

The present invention is possible in any of these shapes. Although it is possible even when the culture dishes are not formed continuously, the practical convenience is low from the above-mentioned point. From the viewpoint of use in cell culture, a material having a transparent material property capable of observing the state of a cell is suitable, but is not particularly limited.

The material of the culture plate is preferably a plastic material from the viewpoint of surface treatment efficiency. However, glass,
It is not limited that it cannot be used for ceramics. Any material that has no toxicity to cultured cells and can be surface-treated can be used.

In order to change the culture area of each culture dish at an arbitrary ratio, a culture area and a non-culture area must be formed. For this purpose, various methods can be applied. As a method for forming a cell culture surface, a method for introducing an oxygen functional group or a nitrogen-containing functional group is preferable. For this purpose, a low-temperature plasma treatment, corona discharge treatment, ultraviolet irradiation, or the like can be used, but a low-temperature plasma treatment method is preferable from the viewpoint of introduction efficiency and the like. A limited culture surface can be formed by coating a part thereof and performing these treatments. By changing the coated surface at any ratio, the culture surface can be changed at any ratio.

However, mere introduction of an oxygen-containing functional group or a nitrogen-containing functional group does not necessarily change the cell culture region arbitrarily. That is, the intended results are not always obtained for all cells by such treatment.

There is also a method in which a cell-adhesive protein or a basic polymer having a cell-adhesive action is previously coated and subjected to the same treatment as described above. In this method, a plasma treatment or the like acts as an action to deactivate the adhesive protein, and the remaining portion (coated portion) becomes a culture region. However, also in this method, the inactivated portion does not show a positive cell non-adhesive action, and a sufficient result cannot be obtained.

[0015] Cell adhesion proteins include collagen,
Basic polymers such as fibronectin, laminin, vitronectin, and the like include polylysine, polyornithine, oliarylamine, and polyallylamine hydrochloride. A method using these mixtures and a coating method such as laminating them are also suitable.

In order to constitute the non-adhesive part of the cell,
Low-temperature plasma treatment in a gas atmosphere containing elemental fluorine is preferable. As a gas containing a fluorine element, tetrafluoromethane, pentafluoroethane, tetrafluoroethylene and the like are preferable. A method of forming and coating a polymer such as Teflon without performing such a surface treatment is also possible, but it is difficult to obtain a high-precision polymer, and there is a problem in transparency. It is preferable to apply a surface treatment to a molded product of transparent plastic, glass, or quartz.

This method can be applied to the case where a cell adhesive protein or the like is used. In this case, simply denaturing the coating protein is not effective enough, and it is necessary to form the fluorine-containing surface by etching effect or plasma polymerization. That is, the cell-adhesive protein is scraped off by an etching effect and a fluorine element is introduced into the substrate surface, or a non-adhesive surface is formed by forming a fluorine-containing thin film by plasma polymerization and covering the cell-adhesive protein. .

A culture dish in which the culture area is continuously changed includes, for example, a culture area of 100%, 75%, 50%,
It means that 5% and 0% culture dishes are continuously formed. This ratio is not particularly limited, and is 50
%, 30%, 10%, or any other ratio. However, as far as cell culture is concerned, this should be 1%
It is not very suitable to take such small intervals. Bioassays using cell culture cannot be expected to be so precise. However, it is not necessarily meaningless if an evaluation system is set up with sufficient allowance for errors.

As a method for forming a culture surface and a non-culture surface separately, such as a low-temperature plasma method, a method of exposing a treatment surface to a space where plasma is generated is used in addition to a method of coating and treating the surface. You can also. Further, it is easy to narrow the irradiation area by forming a beam of ultraviolet light, laser, or the like. Although finer regions can be formed by using a photoresist or the like, ordinary bioassays do not require much accuracy as described above.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples. (Example) (Preparation of culture plate) Culture area ratio 100%, 80%,
Preparation of glue plate with 60%, 40%, 20% and 0% as one series. A 24-well plate (manufactured by Sumitomo Bakelite Co., Ltd., sterilized by gamma ray after molding, inner diameter of the well is 16 mm) formed of polystyrene was arranged in one horizontal row, and six wells (six culture dishes) were arranged in one line, thereby forming four lines in one plate. First, diameter 16mm,
A 1 mm thick silicone piece was subjected to ultrasonic cleaning with ethanol and pure water, and was sufficiently dried. Next, this section is left as it is in the leftmost column, the fan-shaped part with an internal angle of 72 degrees is cut out in the next column, and 144 degrees, 216 degrees, 28
A section from which a sector of 8 degrees was cut was inserted and set in a low-temperature plasma apparatus. Decompressed tetrafluoromethane is introduced.
A power of 100 W was applied at 0.1 Torr, and the treatment was performed for 5 minutes. After a silicon section was taken out of the plate taken out of the apparatus, the portion subjected to the plasma treatment was covered with a new silicon section, and set again in the plasma apparatus. Similarly, nitrogen gas was introduced under reduced pressure. 0.06 Torr, 3 at 50 W
Minutes. After the plasma treatment was completed, the slices were removed, the lid of the plate was attached, and gamma ray sterilization was performed (dose of 10 kGy).

(Preparation of Comparative Culture Plate) Using the same 24-well plate as described above, plasma treatment was performed under a nitrogen gas atmosphere under the same conditions without using silicon slices, and gamma ray sterilization was performed.

(Culture of cells using prepared culture plate) C6 cells, which are glial cell lines, were cultured with 10 fetal bovine serum.
Prepared at 2.0 × 104 c / ml with the DMEM culture solution to which the% amount was added. The cell solution was added at 0.5 ml / well and allowed to stand for 30 minutes. Thereafter, the culture solution was replaced with a new culture solution, and the cells were cultured for 3 days.

(Cell culture using comparative culture plate)
Similarly, C6 cells were 2.0 × 10 4 c / ml (100%), 1.6 to match the ratio of one line on the plate.
× 104c / ml (80%), 1.2 × 104c / ml
(60%), 0.8 × 104 c / ml (40%), 0.
4 × 104 c / ml (20%), culture medium only (0
%), Was prepared. 0.5 ml / well of each cell solution was added and cultured for 3 days.

(Cell ratio after culture) After 3 days, a cell proliferation assay kit (manufactured by Dojindo Laboratories) to measure the number of cells
The resulting WST-1 formazan was measured. Reagents were added at 50 μl / well and incubated for 2 hours at 37 ° C. The solution was transferred to a 96-well plate at 150 μl / well, and the absorbance was measured at a measurement wavelength of 450 nm.
When the X-axis and the Y-axis were plotted by taking the absorbance and the culture area, the plate of the present invention showed good linearity, and a good proportional relationship between the culture area and the number of cells was recognized. Similarly, when the absorbance and the initial cell number were plotted on the comparative plate, no linearity was shown. At a low cell concentration, no proportional relationship was observed.

[0025]

By using the culture plate of the present invention, the number of cultured cells can be arbitrarily controlled without changing the culture environment. This makes it possible to use it for bioassays under various conditions, broadens the application range of pharmacological tests and toxicity tests, and provides high convenience.

Claims (6)

(57) [Claims] 1. A culture plate in which two or more culture plates having the same shape are continuously formed, wherein the culture plate comprises an area for culturing adhesive cells and a non-cultivation area for not culturing adhesive cells. A culture plate, wherein at least two culture dishes have different areas for culturing adherent cells. 2. The culturing region is formed by low-temperature plasma treatment in a gas atmosphere in which an oxygen-containing functional group, a nitrogen-containing functional group, or a mixed functional group thereof can be introduced. A culture plate as described. 3. The culture plate according to claim 1, wherein the region to be cultured is coated with a cell adhesive protein or a basic polymer. 4. The culture plate according to claim 1, wherein the non-culture region is formed by low-temperature plasma treatment in a fluorine-containing gas atmosphere. 5. A culture method comprising culturing adhesion-dependent cells on the culture plate according to claim 1, 2, 3, or 4. 6. A test method using a cell cultured on the culture plate according to claim 1, 2, 3, or 4 for a bioassay.