WO2003038031A1

WO2003038031A1 - Cell culturing system and method for measuring dose response

Info

Publication number: WO2003038031A1
Application number: PCT/JP2002/011339
Authority: WO
Inventors: Kazuo Ohsaki; Akikuni Hara
Original assignee: Hakuju Institute For Health Science Co., Ltd.
Priority date: 2001-10-31
Filing date: 2002-10-31
Publication date: 2003-05-08
Also published as: NO20032988L; NO20032988D0; TW200300450A; JP2005102501A

Abstract

A cell culturing system comprising a container (16) for culturing cells in a culture medium by applying a specified voltage between a pair of electrodes (18) disposed at the opposite ends thereby conducting a current between the electrodes, and current density generating means (23, 25, 27) for imparting at least two different current densities to the cells in the culture medium within the container, wherein the current density generating means are arranged in series with the flowing current system. Dose response of the cells by a different quantity of current can thereby be measured accurately even if the timing of experiment is divided and the properties of cells being injected are different.

Description

Description Cell culture device and method for measuring dose response

The present invention relates to a cell culture device for culturing cells in a medium and a method for measuring dose responsiveness. Background art

In general, in order to measure the dose response of a cell due to a direct current or an induced current, a different amount of current is applied to the cell, or a different electric field is applied to the cell, and an electric field is applied to induce a different amount of induced current and the cell is dosed. The difference in responsiveness to is measured. Responsiveness includes changes in cell number, changes in cell genes, changes in cell chromosomes, changes in cell functions, changes in cell components, and the like. In general, culture dishes, culture cages, and culture membranes are used as the cell retention system to examine responsiveness, but culture dishes of the same area, culture cages of the same area, and culture membranes of the same area are used. Have been. The cells are usually cultured in a culture dish or the like, and the cells taken out therefrom are diluted, and a certain amount is collected and put into a cell holding system. This means that the cells are placed in the retention system each time different dose experiments are performed. Then, different currents are applied at different times to measure the change.

In the above method, the timing of the experiment is divided for each different dose. Unless the cells are at the same time, it is impossible to confirm 100% of the properties and cell number of the cells to be compared for dose. This is because over time, cultured cells have the potential to proliferate and change their properties under ambient conditions. Therefore, it is necessary to measure the responsiveness of cells due to doses of different cells, and inevitably is inaccurate. Therefore, conventionally, in order to eliminate the above-mentioned disadvantages, many experimental instruments were used to supply different amounts of electricity, and the same cells held in many holding systems at the same time were incorporated into the experimental instruments. It was required to start the experiment at the time.

The present invention has been made in view of the above-described drawbacks, and an object of the present invention is to provide a method in which the timing of an experiment is divided and the properties of cells to be injected are different, and the amount of current is different even if the number of cells is different. It is an object of the present invention to provide a cell culture device and a dose response measuring method capable of accurately measuring the dose response of cells. Disclosure of the invention

A cell culture device according to the present invention includes a container for applying a predetermined voltage between a pair of electrodes provided at opposite ends and flowing a current between the electrodes to culture cells in a culture medium; Current density generating means for giving at least two different current densities to cells in a medium in the container, wherein the current density generating means is arranged in series with the flowing current system.

A cell culture device according to the present invention includes a container for applying a predetermined voltage between a pair of electrodes provided at opposite ends and flowing a current between the electrodes to culture cells in a culture medium; Current density generating means for providing at least two different current densities to cells in a culture medium in the container, the current density generating means being disposed in series with the flowing current system, An electro-permeable barrier member is provided between the generating means and the electrode.

In the cell culture apparatus according to the present invention, the current density generating means is configured by a permeable main brain membrane having an ultra-micro structure, and a plurality of the main brain membranes having different areas have a predetermined area. It is characterized by being provided on holders arranged at intervals.

The dose responsiveness measuring method according to the present invention includes: a step of applying a predetermined voltage between a pair of electrodes provided at opposite ends and causing a current to flow between the electrodes; Providing two different current densities. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing a cell culture device according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an embodiment of the cell culture device according to the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is an outline of a cell culture device. Reference numeral 10 indicates a cell culture device. The cell culture device includes a power supply 12 and a current control device 14 for controlling a current from the power supply. This is connected in series to the electrode of the cell culture device, and a predetermined amount of current flows through the closed circuit. The cell culture apparatus 10 includes a container 16 having a rectangular parallelepiped internal hollow predetermined volume and capable of holding a fluid. The container is provided with electrodes 18 and 18 on opposite ends (inner walls), respectively. Then, a medium for measuring the responsiveness of the cells is placed in the container. In the container 16, a plurality of pairs, for example, three pairs of holders 22, 24, and 26 are disposed at a predetermined interval between the two electrodes. The spacing between the holders may be arbitrary. The plurality of holders are respectively arranged in series toward the system through which the current flows. The carrier is made of glass or plastic.

The holders 22, 24, and 26 are provided with a transparent main brain membrane 23, 25, 27 having an ultramicro structure that has good current permeability and does not allow cells to enter and exit. The permeability of the mainbrene membrane is the mesh diameter of its mesh; about 0.3 mm, the thickness: several to about 10 microns.

Then, an electro-permeable barrier member 28, 28, such as agarose, is disposed between the container and the holding member. The agarose may be applied to the surface of each electrode. By providing agarose, it is possible to reduce the influence on the cells of the chemical reaction near the electrode surface.

The holders 22, 24, and 26 are provided with the permeable main brain films 23, 25, and 27 only in the portion where current flows. For example, a substantially circular permeable main brain film is provided only at a predetermined position where a current flows between both electrodes. The diameters d1, d2, and d3 are set so that the circular areas of the permeable main brain membranes 23, 25, and 27 are different for each of the three pairs of holders. Assuming that the circular area ratio of the permeable main brain film is 1: 2: 3, the current densities are 3: 2: 1, respectively.

A culture medium containing cells is put between a pair of holding members provided at a predetermined interval, and an electro-permeable barrier such as agarose is provided between the container and the holding member. Thereafter, a predetermined voltage is applied between both electrodes to flow a current. The current density in the medium is obtained by dividing the value of the applied current by the area of the permeable main brain membrane. A predetermined voltage is applied between a pair of electrodes provided at opposite ends to flow a current between the electrodes. At the same time, at least two different current densities are given to cells in the culture medium to improve dose responsiveness. Measure.

In the above embodiment, a pair of electrodes are arranged in the left-right direction so that the current flows in a horizontal direction. However, a pair of electrodes may be arranged in the vertical direction so that the current flows in the vertical direction. By flowing the current in the vertical direction, it is possible to more stably and uniformly flow the current to the cells.

After passing the current for a certain period of time, the holder is pulled up and the cells are used for responsiveness evaluation. Therefore, in the same cell at the same time, cell responsiveness with different current densities can be obtained. The shape of the film is not limited to a circle but may be a rectangle. The permeable main brain membrane attaches the cells. Cells are cells having adsorbability, such as endothelial cells and fibroblasts.

ADVANTAGE OF THE INVENTION According to the cell culture apparatus and the dose-response measuring method concerning this invention, the time of an experiment is divided | segmented. Can be measured. Industrial applicability

A cell culture device for culturing cells in a medium and a method for measuring dose responsiveness.

Claims

The scope of the claims

1. A container for applying a predetermined voltage between a pair of electrodes provided at opposite ends and flowing a current between the electrodes to culture cells in the medium, and cells in the medium in the container. Further comprising current density generating means for providing at least two different current densities to the cell culture apparatus, wherein the current density generating means is arranged in series with the flowing current system.

2. A container for applying a predetermined voltage between a pair of electrodes provided at opposite ends and flowing a current between the electrodes to culture cells in the culture medium, and cells in the culture medium in the container. Current density generating means for giving at least two different current densities to the current system, wherein the current density generating means is arranged in series with the flowing current system, and the current density generating means and the electrode An electrically permeable barrier member disposed therebetween.

3. The current density generating means is constituted by a permeable main brain membrane having an ultra-micro structure through which cells cannot enter and exit, and a plurality of the main brain membranes having different areas are held at predetermined intervals. 3. The cell culture device according to claim 1, wherein the cell culture device is provided on a body.

4. applying a predetermined voltage between a pair of electrodes provided at opposite ends to flow a current between the electrodes;

Applying at least two different current densities to the cells in the medium at the same time.