JP2006333861A - Method for electrically stimulating cultured muscular cell by using transparent electrode - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide apparatus for culturing cells on a transparent conductive electrode and imparts uniform electrical stimulations on the cells and enabling an optical observation on the live cells and easy to achieve a high throughput. <P>SOLUTION: The invention relates to the apparatus for culturing live cells on the transparent conductive plate, uniformly imparting the electrical stimulation to the live cells on a transparent plate by loading a voltage between the transparent conductive plate and an upper electrode, and optically observing the live cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culture method that makes it possible to apply electrical stimulation to cultured muscle cells uniformly by culturing cultured muscle cells on an electrically conductive transparent plate.

  Muscle cells (skeletal muscle / myocardium) can be stimulated electrically, and in the field of cell biology, analysis of the response of these cells to electrical stimulation is frequently performed. Conventionally, as a method for electrically stimulating cells, a method of applying an electric field parallel to cells adhered to the bottom surface is generally used. Sold. However, such an arrangement has drawbacks that it is difficult to screen for many culture and electrical stimulation conditions and that the electric field is not applied to the cells uniformly.

  In order to solve the above-mentioned problems in the cultured muscle cell electrical stimulation method, methods for applying electrical stimulation to cells from the up and down directions as in Patent Document 1 and Patent Document 2 have been devised. However, these methods make the most important experiment in cell biology of optical observation of cells impossible because the lower electrode is opaque. Moreover, since it is necessary to wire each chamber, it is difficult to say that it is suitable for screening of electrical stimulation conditions.

Patent Publication 2003-225 Patent Publication 2002-335945 The C-Pace 100-Culture Paper and C-Dish 100-Culture Disc electrode; IonOptix Corporation Internet <URL: http: // www. ionoptix. com />

  As described above, it is difficult to apply electrical stimulation to cultured muscle cells by a method suitable for screening of drugs and electrical stimulation conditions by the conventional technique in which electrodes are arranged in parallel. In addition, the method in which the electrodes are arranged in the vertical direction has a drawback that optical observation cannot be performed because cells are cultured on an opaque permeable membrane.

  An object of the present invention is to provide a device that cultures cultured muscle cells on an electrically conductive transparent electrode, uniformly applies electrical stimulation to the cultured muscle cells, enables optical observation, and facilitates high throughput.

  According to the present invention, cultured muscle cells are cultured on an electrically conductive transparent plate, and a voltage is applied between the electrically conductive transparent plate and the upper electrode to uniformly stimulate the cultured muscle cells on the transparent plate. Provided is a device which can be optically observed while the cells are alive.

  In addition, by using the cellular electrical stimulation device of the present invention, it is possible to easily perform screening for drugs and electrical stimulation conditions.

  Further, the present invention provides a container having a predetermined volume formed by placing an optically transparent electrode on the bottom, and the container is sealed from above the container, and disposed opposite to the transparent electrode. There is provided an electrical stimulation cuvette comprising a counter electrode, and comprising a lid body disposed opposite to the container in the vertical direction.

  As described above, according to the present invention, it is possible to apply electrical stimulation to cultured muscle cells cultured on an electrically conductive transparent plate and simultaneously optically observe the cells. In the field of cultured muscle cell research, it is important to examine the electrical stimulus-dependent response of cells, and optical observation is one of the main means. Further, since the electrodes are arranged in the vertical direction, they are suitable for screening for drugs and electrical stimulation conditions. Furthermore, since the electrically conductive transparent plate is obtained by vapor-depositing an optically transparent electrically conductive material on the glass surface, a circuit is formed on the glass surface so that various types can be simultaneously performed on the same glass surface depending on the part. It is possible to give electrical stimulation under various conditions. In addition, by applying electrical stimulation to the cultured muscle cells, it is possible to give a pseudo-motion effect to the cells, so that it is possible to investigate the influence of the drug on the intracellular energy metabolism.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram showing a schematic configuration of a cultured muscle cell electrical stimulation device according to an embodiment of the present invention, and FIG. 2 is an external perspective view thereof. Referring to FIG. 1, an electrically conductive transparent plate (1) is placed at the bottom of a well-type culture dish (4) so that the electrically conductive surface faces upward, and cultured muscle cells are placed on the electrically conductive transparent plate. Incubate (3). The upper electrode (5) is placed on the lid (6) so as to be in contact with the culture medium (2), and the cultured muscle cells (3) are electrically connected by energizing between the electrically conductive transparent plates (1) placed on the lower part. Give a stimulus.

  In this example, C2C12 myoblasts induced to differentiate into muscle-like tissue in a low nutrient medium were used. This cell expresses a voltage-dependent channel after differentiation induction, and contraction movement occurs by electrical stimulation. Therefore, the stimulation according to the present invention is the optimal cell. C2C12 myoblasts were cultured in Dulbecco's modified Egel medium + 10% fetal bovine serum on a glass substrate coated with ZnO (FIG. 3). After culturing under the above conditions for 3 days, the C2C12 myotube cells differentiated were obtained by substituting Dulbecco's modified Egel medium + 2% bovine serum and culturing for 1 week (FIG. 4). It can be seen that the cells are differentiated from each other because the cells are fused and show the structure on the tube.

  Electrical stimulation was applied to the differentiated C2C12 cells cultured on a glass substrate coated with ZnO by the apparatus according to the present invention. Since C2C12 cells differentiate into muscle-like tissues, contraction can be induced by electrical stimulation. When C-Path of IonOptix was used as the electrical stimulation generator and electrical stimulation was applied at a voltage of 5 V, a period of 1 Hz, and a pulse width of 24 ms, cell contraction was observed (FIG. 5). When the change in the brightness of a specific part of the phase contrast image was examined, it can be seen that the cells contracted in response to the applied electrical stimulation since they periodically change at 1 Hz (FIG. 6).

  The muscle cell electrical stimulation apparatus according to the present invention can be applied to various fields such as molecular biology, cell biology, genetic engineering, and regenerative medicine in muscle cells.

1 is a schematic configuration of an electrical stimulation device according to an embodiment of the present invention. 1 is an external perspective view of an electrical stimulation device according to an embodiment of the present invention. C2C12 cells before differentiation induction cultured on a glass substrate coated with ZnO. C2C12 cells after differentiation induction cultured on a glass substrate coated with ZnO. Electrical stimulation was applied to the differentiated C2C12 cells cultured on a ZnO-coated glass substrate by the method of the present invention. The right figure is an enlarged view of the part surrounded by the white square in the left figure. The upper right is before electrical stimulation. The lower right is after electrical stimulation. It can be seen that the cells are contracted by electrical stimulation and the striped pattern moves (see arrow). A cell that responds to periodic electrical stimulation (voltage 5 V, period 1 Hz, pulse width 24 ms). The change over time of the brightness of the part surrounded by the white square in the left figure was examined (right figure). It can be seen that the brightness changes in response to electrical stimulation as the cells move.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Electrically conductive transparent plate 2 Medium 3 Cultured muscle cell 4 Well-type culture dish 5 Upper electrode 6 Lid

Claims (4)

Cell culture characterized by culturing cultured muscle cells on an electrically conductive transparent plate and applying electrical stimulation to the cultured muscle cells on the transparent plate by applying a voltage between the electrically conductive transparent plate and the electrode Law. A cell culture method characterized in that the electrically conductive transparent plate of the previous period is a product obtained by depositing a ZnO film on a glass surface. A cell culture method characterized in that various electrical stimulation patterns are given to cultured muscle cells by creating a pattern circuit on a ZnO film on an electrically conductive transparent plate in the previous period. A container having a predetermined volume formed by installing an optically transparent electrode at the bottom, and a counter electrode disposed over the container to seal the container and to face the transparent electrode, The cuvette for electrical stimulation according to claim 3, further comprising a container and a lid body disposed to face the container in the vertical direction.