JPS63283584A - Electric stimulation of cell - Google Patents

Abstract

PURPOSE:To raise cell fusion efficiency, by applying a high-frequency electric field, a low-voltage DC pulse electric field, a high-voltage DC pulse electric field, a low-voltage DC pulse electric field and a high-frequency electric field in this order to cell suspension placed between electrodes. CONSTITUTION:A changeover switch 6 is changed to a contact (a) and a high-frequency electric field having about 100-400V/cm field strength at 1MHz is applied from a high-frequency generating part 1 to cells between electrodes 5a and 5b. By the application, cell A is brought into contact with cell B. Then the changeover switch is changed to a contact (b) by a signal from a control part 4. When a low-voltage DC pulse electric field is impressed from a low- voltage pulse generating part 2 to the cells, the contact parts between the cells are extended from points to a plane. Then the changeover switch 6 is changed to a contact (c) by a signal from the control part 4. When a high-voltage DC pulse electric field is impressed from a high-voltage pulse generating part 3 to the cells, the cells are mutually fused.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a cell electrical stimulation method for applying electrical stimulation to cells suspended in a solvent to fuse the cells.

(Prior Art) One method for fusing cells is to use electrical stimulation. In the cell electrical stimulation method, a cell suspension in which cells are suspended at an appropriate density is placed between a pair of opposing electrodes, and electrical stimulation is applied to the cells from the electrodes. First, the electrical stimulation conditions are approximately 100 to 4 MHz at about IMHz.
A high-frequency electric field of about 0.00 V/am is applied, and then a high-voltage DC pulsed electric field with a constant pulse width is applied one to several times.

First, a high-frequency electric field is applied to bring the cells into contact according to the principle of dielectric electrophoresis, and then a high-voltage DC pulsed electric field is applied to disrupt the cell membrane and increase its permeability. This is to fuse the cells and the cells.

When a high-voltage DC pulsed electric field is applied to a cell, the cell membrane is disturbed, and theoretical considerations have been made as to how much electric field should be applied to make the cell membrane permeable.The required electric field strength E (KV/ Cm) can be calculated. That is, E,=2Vm·cose/3r. Here, V+s is the potential difference acting on the cell membrane.

r is the radius of the cell, θ is the angle between the direction of the electric field and a point on the cell surface, and when the electric field strength E is applied so that V+n is about 1 V, the cell membrane becomes permeable.

(Problems to be Solved by the Invention) In order to increase the efficiency of cell fusion by electrical stimulation, it is necessary that the cells are in strong contact with each other at the time of applying the high voltage DC pulsed electric field. In order to strengthen the contact between cells and cells using a high-frequency electric field, the voltage must be increased, but because the frequency is high, it is extremely difficult to increase the voltage and power.

In order to increase cell-to-cell contact, methods are being considered that use centrifugal force to bring cells into close contact with each other, but this method requires mechanical manipulation, resulting in a lack of reproducibility.

The present invention aims to increase cell-to-cell contact and increase cell fusion efficiency without increasing the voltage of a high-frequency electric field or requiring mechanical operations such as centrifugal force.

(Means for solving the problem) In the present invention, a cell suspension in which cells are suspended in a solvent is placed between a pair of electrodes, and a high-frequency electric field, a low-voltage DC pulsed electric field, and a high-voltage DC A pulsed electric field, a low-voltage DC pulsed electric field, and a high-frequency electric field are applied in this order.

(Example) FIG. 1 shows an example of an apparatus for implementing the present invention, and FIG. 2 is a waveform diagram showing an electric field applied to cells using the apparatus of FIG. 1.

In FIG. 1, 5 is a chamber for performing cell fusion, and is equipped with a pair of parallel electrodes 5a and 5b facing each other, and a cell suspension is formed between these parallel electrodes 5a and 5 and the insulator of the chamber body. It forms a space to accommodate.

A cell suspension 7 is placed between the parallel electrodes 5a and 5b in the space of the chamber 5. For example, the cell suspension 7 contains two types of cells A.
and B are suspended at appropriate concentrations. As a solvent for the cell suspension 7, a solvent to which the osmotic pressure is adjusted with a sugar such as glucose and several mM of CaCf1: or MgCQC can be used can be used.

One electrode 5a of the chamber 5 is connected to a changeover switch 6, and the three contacts a of the changeover switch 6.

A high frequency generator 1, a low voltage pulse generator 2, and a high voltage pulse generator 3 are connected to b and c, respectively. The high frequency generator 1, the low voltage pulse generator 2, and the high voltage pulse generator 3 are each connected to the other electrode 5b of the chamber 5, and by switching the changeover switch 6,
Either the high-frequency generator 1, the low-voltage pulse generator 2, or the high-voltage pulse generator 3 is connected to the electrodes 5a and 5b, and can apply an electric field to the cell suspension 7 between the electrodes 5a and 5b. It looks like this.

Reference numeral 4 denotes a control section, which switches the changeover switch 6 and adjusts the outputs of the high frequency generation section 1, the low voltage pulse generation section 2, and the high voltage pulse generation section 3.

The frequency of the high frequency generator l is IMHz and the electric field strength is 100.
It can output high frequency of ~400V/am. The high-voltage pulse generator 3 generates high-voltage direct current pulses in conventional cell fusion.
A DC pulsed electric field with an electric field strength of ~ several KV/cm can be applied.

The low voltage pulse generator 2 is capable of applying a DC pulse electric field having an electric field strength of about l/s that of the high voltage pulse generator 3.

Next, the operation of one embodiment will be explained.

First, switch the changeover switch 6 to contact a as shown in the figure, and apply the voltage from the high frequency generator 1 to the cells between the electrodes 5a and 5b.
A high frequency electric field with an electric field strength of 100 to 400 V/cm at MHz is applied. The magnitude of the electric field strength depends on the size of the cell; the smaller the cell, the greater the electric field strength required. This is because the strength of the electric field determines the strength of the cell's polarization effect, and the smaller the cell, the smaller its surface charge.
Larger electric field strength is required compared to larger cells. The high frequency electric field is applied for a period of t+ (about 10 seconds) in FIG. 2, and the electric field strength is Vac+ (about 400 V/cm).

The reason for applying a high frequency electric field is to cause polarization only in cells and prevent electrolysis of the solvent.

Application of a high frequency electric field brings cells A and B into contact as shown at the top of FIG.

Next, the changeover switch 6 is switched to the contact position by a signal from the control section 4, and a low voltage DC pulse electric field is applied from the low voltage pulse generating section 2 as shown in a period t2 in FIG. The low voltage DC pulse has an electric field strength Vdc+, a pulse width Pwl, a pulse interval t4, and a repetition number n1. Due to the polarization effect of this DC pulse, the cells that are already in contact come into closer contact as shown in the upper part of FIG. 2, and the junction between the cells is expanded from a point to a plane.

The electric field strength Vdc+ of the low-voltage DC pulse at this time is to the extent that it does not damage the cells, and the purpose is simply to polarize the cells and bring the cells into close contact with each other. Set the voltage to be smaller than the DC pulse voltage.

Next, the changeover switch 6 is switched to the contact point C by a signal from the control section 4, and a high voltage DC pulsed electric field for fusion is applied from the high voltage pulse generation section 3. As shown in FIG. 2, this high voltage DC pulse has an electric field strength of Vd
cz, pulse width PV2, interval t5. The number of repetitions is n2.

As an example of a high voltage DC pulsed electric field, for example, in animal cells, the electric field strength Vdcs is 3 KV/cm, the pulse width Pw2 is 10 to 20 μsec, the pulse interval t5 is 1 second, and the number of repetitions is n.
: is 2 to 3 times. For example, in plant cells, the electric field strength Vdc: is IKV/am, and the pulse width Pv2 is 20 to 30
μ seconds, pulse interval t5 is 1 second, number of repetitions n: is 2 to 3
It is times.

Application of a high-voltage DC pulsed electric field causes the cells to fuse with each other, as shown at the top of FIG. 2.

Thereafter, the changeover switch 6 is switched to the contact position, and the low voltage DC pulse electric field is changed to the electric field strength V as shown in FIG.
dcs, pulse width Pw3, interval t6, and number of repetitions n3, and then the selector switch 6 is switched to contact a, and the high-frequency electric field is applied with the electric field strength Vac as shown in FIG.
:=, apply only for period t3. This completes cell fusion.

The application conditions of the high-frequency electric field and the low-voltage DC pulse applied before and after the high-voltage DC pulse do not need to be very strict, and can be made to be approximately the same before and after the high-voltage DC pulse, for example.

In addition, if the cell density of the cell suspension is increased, a low-voltage DC pulsed electric field can be suddenly applied without applying a high-frequency electric field to bring the cells into close contact with each other.

(Effects of the Invention) In the present invention, before and after applying a high-voltage DC pulsed electric field to increase cell membrane permeability and cause cell fusion, a low-voltage DC pulsed electric field is applied to increase cell-to-cell contact. As a result, the efficiency of cell fusion is higher than in the conventional method of applying a high-voltage DC pulsed electric field immediately after applying a high-frequency electric field.

If we were to increase the fusion efficiency using the conventional method, we would have to increase the output of the high-frequency generator, but such a method is difficult to implement.Therefore, in the present invention, we do not have to increase the output of the high-frequency generator. The device can be simplified.

In the present invention, since it is possible to induce a polarization effect that cannot be achieved only by applying a high-frequency electric field, the fusion efficiency can also be increased in this respect.

In addition, since a low-voltage DC pulsed electric field is applied to stimulate the cell membrane before applying a high-voltage DC pulsed electric field, the strength of the high-voltage DC pulsed electric field is reduced and the survival rate of cells can be increased. .

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing an example of an apparatus for implementing the present invention, and FIG. 2 is a waveform diagram showing one embodiment together with a fused state. l... High frequency generation section, 2... Low voltage pulse generation section, 3... High voltage pulse generation section, 4... Control section, 5... ...Chamber, 5a, 5b... Electrode, 6... Changeover switch. 7...Cell suspension.

Claims (1)

[Claims] (1) A cell suspension in which cells are suspended in a solvent is placed between a pair of electrodes, and from the electrodes the cells are exposed to a high frequency electric field, a low voltage DC pulsed electric field, a high voltage DC pulsed electric field, a low voltage DC pulsed electric field. A cell electrical stimulation method that applies high-frequency electric fields in this order.