JPH074217B2 - Power supply for electric cell fusion - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a cell fusion power source used for inducing cells by an electric field and an electric stimulus, and fusing cells or providing micropores in cells (electroporation). .

Conventionally, cell fusion by the dielectrophoresis method, in which cells are arranged in a beaded array on an electrode using an electric field, is performed by using PEG (polyethylene glycol), Sendai virus, etc. Compared to cell fusion performed, the operability is good, and since the medium used is not toxic to cells, washing of cells immediately after fusion is unnecessary, and it has recently attracted attention as an extremely simple and reliable method. In this case, the range of applications is extremely wide, such as electroporation for making holes in the cytoplasmic membrane.

However, in order to generate the dielectrophoretic force, a power supply that outputs an AC voltage of 100 Hz to 100 MHz is required depending on the size and type of cells, so a power supply configuration that uses that wide frequency band is extremely important. It is complicated and the power supply itself becomes expensive.

Further, in the conventional method, by applying a rectangular wave pulse of about 50 to 150 V / cm, the cell membrane in the adjacent portion of a pair of adjacent cells arranged between the electrodes is destroyed, and in fact 1 B. hybrid or allogeneic cells are produced. However, at the same time, a phenomenon occurs in which the cells and the electrodes are bound together, and when taking out a single cell that has been fused from the fusion space, it is possible to detach the cells from the electrode by a mechanical operation such as water flow. This process, if any, is often damaging to cells, contributing to reduced fusion efficiency.

On the other hand, if the electrode surface is coated with a polymer film such as Teflon or silicon that is difficult for cells to adhere to, it is possible to prevent the adhesion of cells, but since these films do not have conductivity, It becomes difficult to apply a pulse voltage through the membrane.

In view of the above, the present inventors have set the stimulation pulse at 100 Hz to 100 MHz.
The electric cell fusion device provided with the stimulation pulse modulation unit that is modulated by means not only solves the problem of cells adhering to the electrodes and improves the stability and operability of fusion, but also simplifies the configuration of the power supply. The present invention has been discovered and has reached the present invention.

Hereinafter, examples of the electric cell fusion device of the present invention will be described in detail.

The reference oscillating unit (11) is a unit for performing accurate pulse oscillation using a crystal oscillator or the like. The dielectrophoresis output section (13)
The oscillation output of the reference oscillating unit (11) is divided and converted into a pulse signal converted into a predetermined frequency. Fusion signal output unit (12)
When an operation signal St is input from the outside, the counter counts the oscillation output of the reference oscillation section (11) and outputs a pulse signal having a predetermined time width. A stimulation pulse modulator (14) divides the output pulse voltage of the fusion signal output unit (12) by a frequency divider circuit or the like at a frequency between 100 Hz and 100 MHz and outputs a modulated pulse voltage.

The fusion signal output unit (12) and the stimulation pulse modulation unit (14)
In the rotation configuration, not only is it configured separately as shown in the block diagram of FIG.
One circuit using a converter that divides the oscillation output from 1) at a frequency between 100Hz and 100MHz and uses the divided output as a pulse voltage with a predetermined time and a predetermined voltage value. You may put together as a structure.

The voltage conversion units (15) and (16) are dielectrophoresis output units (13) and
And the pulse signal output from the fusion signal output section (12) is converted into a voltage pulse having a predetermined voltage value.

FIG. 3 shows an embodiment using the electric cell fusion device of the present invention shown in FIG. In the figure, (I) is the apparatus shown in FIG. Reference numeral 2 is an electrode pair made of a conductive material such as platinum. First
The output end of the figure is connected to the electrode pair in FIG.
The fusion chamber and cell suspension were omitted.

The superimposing section (17) superimposes the outputs of the voltage converting sections (15) and (16) to form an output waveform shown in FIG. 2 (b).

Next, the operation of the electric cell fusion device of the present invention having the above configuration will be described in detail with reference to the drawings.

The output from the reference oscillating unit (11) is divided into two and input to the dielectrophoresis output unit (13) and the fusion signal output unit (12), respectively. The fusion voltage output section (12) is stopped until an external operation start signal St arrives.

In the dielectrophoresis output section (13), the output from the reference oscillation section (11) is frequency-divided and converted into a pulse signal of 100 Hz to 100 MHz, and a pulse having a predetermined voltage value in the voltage conversion section (16). The signal is output to the electrode pair (2) shown in FIG. A dielectrophoretic force is generated by the pulse voltage between the electrode pair (43), and cells A and B are arranged in a beaded pattern between the electrodes as shown in FIG. 4 (a).

After arranging in a beaded shape, the operation start symbol St from the outside is input to the fusion voltage output unit (12), and the fusion voltage output unit (12)
Starts its operation and counts the output from the reference oscillator (11) until it reaches a preset count value. After reaching the count value, a pulse signal having a pulse width corresponding to the count value is output from the fusion voltage output section (12). This pulse signal is 100 Hz to 100 at the stimulation pulse modulator (14).
After being modulated to a predetermined frequency between MHz and converted to a predetermined voltage by the voltage conversion section (15), it is output to the electrode (43) via the superposition section (17). The cells arranged in a beaded pattern between the electrodes (43) are applied with this pulse voltage, and fusion occurs as shown in FIG. 4 (b).

This pulse voltage is a modulated pulse that is a single pulse, which is a conventional fusion pulse voltage, modulated at a predetermined frequency between 100 Hz and 100 MHz, so an electrode whose surface is coated with a polymer film such as Teflon or silicon is used. In the case that the cell fusion occurs, it is possible to induce cell fusion. Therefore, in combination with this electrode, a cell fusion device in which cells do not adhere to the electrode can be formed.

Further, since the elements constituting each block can be easily constituted by a crystal oscillator, a counter, and a frequency divider, the effect is great such that the cells do not adhere to the electrodes with a simple constitution.

[Brief description of drawings]

FIG. 1 is a block diagram showing an embodiment of the present invention, FIG. 2 is an output waveform diagram, FIG. 3 is a diagram showing an embodiment of the present invention, and FIG. 4 is a diagram showing the progress of cell fusion. . (11) …… Reference oscillator, (12) …… Fused signal output, (13) …… Dielectrophoresis output, (14) …… Stimulation pulse modulator, (15) (16) …… Voltage converter , 2 ... electrodes.

Claims (1)

[Claims] 1. A dielectrophoresis signal output means for generating a pulse signal for producing a dielectrophoretic force, a fusion pulse signal output means for setting a pulse width of a stimulation pulse for producing cell fusion, and the fusion pulse. The output pulse of the signal output means
A power supply for electric cell fusion, comprising a modulated pulse signal output means for modulating at 100 Hz to 100 MHz.