JP2006197872A - Method of electroporation and cuvette for electroporation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device capable of performing electroporation of a cell in a state of bonding it on a scaffold and also observing the cell in its living state. <P>SOLUTION: This method for performing the electroporation of the cell is provided by culturing a living body cell on a transparent electrode, and introducing a foreign material such as a gene, etc., into the living cell on the transparent electrode by giving an electric pulse between the transparent electrode and a counter electrode. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell electroporation apparatus that performs electroporation while living cells are adhered to a scaffold.

  Conventionally, when introducing an external substance into a cell during genetic manipulation of the cell, it is necessary to make the cell membrane temporarily permeable, and a lipofection method or an electroporation method has been used. Among these, the lipofection method is toxic to the drug used for lipofection and has a drawback that it cannot be used depending on the cell type.

  The electroporation method is, for example, a method described in Non-Patent Document 1, wherein a cell is peeled off from a scaffold, a microscopic opening is formed in the cell by applying a relatively strong electric field to the cell in a floating state, and an external substance is introduced. However, since the cells need to be in a floating state, there is a drawback that they may not be applicable to cells that are sufficiently differentiated cells or cells forming tissues. In addition, even when electroporation is performed on undifferentiated cells, it must adhere to the scaffold again to induce differentiation, but the effect of the gene introduced by electroporation disappears in about 48 hours, and the cells are sufficiently It was difficult to see the effect of the transgene in a differentiated state.

  In order to solve the above problems in the electroporation method, an electrode in the form of direct contact with a living body as in Patent Document 1 and a needle-shaped electrode as in Patent Document 2 are disclosed. However, these methods have been devised for the purpose of gene introduction into tissues, and there is a problem that cells cannot be observed alive after introduction of an external substance.

  There is a microinjection method as a third gene introduction method. This is a method in which a hollow glass microneedle is inserted into a cell and a foreign substance is directly introduced. However, since it is introduced manually into each cell, it can be introduced even by a highly skilled person. The number of cells was very small.

Japanese translation of PCT publication No. 2002-532166 Special table 2003-505114 gazette Protein, Nucleic Acid, Enzyme, Vol. 31, pp. 1591 to 1603 (1986)

  As described above, the conventional technique has a problem that it is difficult to observe a cell while it is alive by introducing an external substance such as a gene while the cultured cell is adhered to the scaffold.

  An object of the present invention is to provide an apparatus capable of performing electroporation while adhering a cell to a scaffold and observing the cell while alive.

  According to the present invention, an external substance such as a gene can be introduced into a living cell on the transparent electrode by culturing the living cell on the transparent electrode and applying an electric pulse between the transparent electrode and the counter electrode.

  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 electroporation 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, an external substance such as a gene can be introduced by electroporation while the living cells cultured on the transparent electrode are adhered to the scaffold. Further, since the cell adhesion surface is a transparent plate, it is possible to optically observe the morphology of the living cells. In the field of cell biology, the main method is to investigate how the introduced external substance behaves in the differentiated cells and where it is localized, and the cells remain alive. The fact that optical observation is possible is a great advantage. Due to the above characteristics, this electroporation apparatus is useful for introducing an external substance in a situation where the cells die by being peeled off from the scaffold such as after differentiation induction of the cells. In addition, the method of introducing an external substance using the apparatus obtained by the present invention does not require any special technique and has an advantage that anyone can easily perform experiments with high reproducibility.

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

  FIG. 1 is a diagram showing a schematic configuration of an electroporation apparatus according to an embodiment of the present invention. Referring to FIG. 1, a transparent electrode (2) is installed at the lower part of the container (1) so that the electrically conductive surface faces upward, and living cells (4) are cultured on the transparent electrode.

  A transparent electrode outer peripheral part is arrange | positioned so that a metal electrode plate (3) installed in the lower part of a container may contact | connect an upper surface. With such a configuration, the culture solution inside the container can be energized from the lower electrode (6) connected to the electric pulse generator without contacting the metal electrode.

  The counter electrode (5) is placed on the lid (7) and is used when electroporation is given to living cells, and can be cultured in a lid without electrodes during normal culture. During electroporation, the space between the transparent electrode (2) and the counter electrode (5) is filled with a solution containing an external substance to be introduced into the living cell, and is temporarily transmitted to the living cell on the transparent electrode by applying an electric pulse. Gives sex and introduces external substances into living cells.

  In this example, C2C12 myoblasts induced to differentiate into muscle-like tissues in a low nutrient medium were used. This cell is difficult to introduce a gene by lipofection, and if it is peeled off from the substrate after differentiation induction, the method according to the present invention is the optimum gene introduction method because the electroporation method cannot be used. 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.

  The CFP gene was introduced into the differentiated C2C12 cells cultured on a glass substrate coated with ZnO by the apparatus according to the present invention. GenePulsar Xcell of Bio-Rad was used as an electrical pulse generator, and an exponential decay pulse with a voltage of 150 V and a capacitance of 1000 mF was applied in PBS with a DNA concentration of 100 mg / ml at an electrode interval of 3 mm. Thereafter, the cells were cultured in Dulbecco's modified Egel medium + 2% bovine serum for 24 hours, and the fluorescence of CFP was observed with a fluorescence microscope (FIG. 5). Since fluorescent cells (cells that look white in FIG. 5) were observed, the device according to the present invention introduced the CFP gene with the cells adhered to the scaffold, and the gene was expressed in the cells. I understand that.

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

1 is a schematic configuration of an electroporation cuvette according to an embodiment of the present invention. 1 is an external perspective view of an electroporation cuvette according to an embodiment of the present invention. C2C12 cells before differentiation induction cultured on ZnO-coated glass substrate Differentiation-induced C2C12 cells cultured on ZnO-coated glass substrates The fluorescence microscope image of the C2C12 cell which introduce | transduced CFP fluorescent protein by the method by this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container 2 Transparent electrode 3 Metal electrode plate 4 Living cell 5 Counter electrode 6 Lower electrode 7 Lid

Claims (3)

  An electroporation method comprising culturing cells on an electrode of an electroporation cuvette and introducing an external substance into the cell with the cells attached to the electrode surface.   An electroporation cuvette for use in an electroporation method in which cells are cultured on an electrode of an electroporation cuvette, and an external substance is introduced into the cell with the cells attached to the electrode surface, and one electrode of the cuvette A cuvette for electroporation, characterized in that is an optically transparent electrode.   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 electroporation cuvette according to claim 2, further comprising a container and a lid body arranged to face in the vertical direction.