JPH02131584A - Method and device for electrically boring cell - Google Patents

Abstract

PURPOSE:To form a small hole having same size in same position of cell membrane of each cell in controlled state by dispersing a cell into a medium liquid, applying alternating current electric field to arrange the cell in electric field of alternating current electric field and applying direct current pulse electric field in the direction crossing with the electric field direction. CONSTITUTION:A medium liquid 2 containing a cell 1 is put in recessed part 11 of substrate and a switch S1 is turned on and alternating current electric field is applied to a medium liquid 2 from electrodes 3A and 3B to arrange the cell 1 in the direction of electric field of alternating current. Then a switch S2 is turned on and direct current pulse electric field is applied from electrodes 6A and 6B to form small hole 4 in a cell membrane of each cell 1. DNA, etc., in a medium liquid 2 is taken in the cell 1 through the small hole 4 and then switch S2 is turned off to release application of direct current pulse electric field and small hole 4 of cell are gradually repaired. Thereby DNA, etc., can be taken in a number of cells in same conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cell electroporation method in which small pores are formed in a cell membrane using a DC pulsed electric field, and an apparatus used for the method.

[Conventional technology]

With the progress of biotechnology, it has become important to form small pores in the cell membrane of cells and allow DNA and the like to be taken in therein.

It is known that when an alternating current electric field is applied to a medium containing cells, the cells line up in a line in the direction of the electric field (pearl chain phenomenon). As shown in FIG. 4(a), electrodes 3A and 3B are provided for applying an electric field to the medium 2 containing the cells 1.

Then, when an AC voltage v1 is applied between the electrodes 3A and 3B, the cells 1 are arranged in a crowbar chain shape in the direction of the electric field. Therefore, by turning on the switch Sl, the electrode 3
When a DC pulse voltage 2 is applied between A and 3B, a small hole 4 is formed in the cell 1 as shown in FIG. 4(b).

However, since the small holes 4 formed in the cell membrane of the cells 1 are located in the direction of the electric field, the adjacent cells 1 fuse with each other through the small holes 4 after the application of the DC pulse voltage v2 is removed. , resulting in giant cells. For this purpose, individual cells 1 are electroporated in a separated state, and DNA is injected into the cells 1.
difficult to incorporate. Therefore, when DNA or the like is to be taken in, a small pore is formed in the cell 1 as shown in FIG. 5.

As shown in FIG. 5(a), a pair of electrodes 3A and 3B are placed in a medium 2 containing cells 1.

Then, when the switch Sl is turned on and the DC pulse voltage V2 is applied as shown in FIG. 2(b), the cell membrane of the cell 1 is electroporated. At this time, if DNA is included in the medium 2, the DNA can be taken into the cell 1 through the electrically formed small pores 4. Then, by turning off the switch Sl and canceling the application of the DC pulse voltage 2, the small pore 4 formed in the cell 1 is repaired, and as a result, the cell 1 that has taken in the DNA is obtained.

[Problem to be solved by the invention]

However, in the method shown in FIG. 5, the positions at which the small holes 4 are formed are not constant for each cell, and it is also difficult to form holes in all cells. For this reason, DNA uptake and the like could not be carried out with good control in all cells.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a cell electroporation method capable of forming small pores with good controllability in cells in a medium, and an apparatus used therefor.

[Means to solve the problem]

The cell electroporation method according to the present invention includes a first step in which an alternating current electric field is applied to a medium solution to arrange cells in the medium solution in the electric field direction of the alternating current electric field, and a direct current is applied in a direction crossing the electric field direction of the alternating current electric field. A second step of forming small pores in the cell membrane of the cell by applying a pulsed electric field. Here, a substance such as DNA may be included in the medium in advance.

Further, the cell electroporation device according to the present invention includes a base body having a recess formed therein for containing a medium containing cells, and a pair of base bodies disposed on the base so as to be able to apply an electric field to the medium placed in the recess. a first electrode, a pair of second electrodes disposed on the base so as to be able to apply an electric field to the medium in a direction crossing the electric field direction of the electric field generated by the pair of first electrodes; AC power supply means for supplying an alternating current voltage for arranging the cells, and a direct current pulse supplying a direct current pulse voltage for forming small pores in the cell membrane to a second electrode after supplying the alternating voltage to the first electrode. It is characterized by comprising a power source means. Here, the second electrode may be composed of a plurality of electrode members, and the plurality of electrode members may be electrically isolated from each other until just before the application of the DC pulse voltage.

[Effect]

According to the cell electroporation method of the present invention, cells are arranged in a fixed direction by an alternating current electric field (burl chain phenomenon), and the cell membranes of these cells are perforated by a direct current pulsed electric field in a direction that intersects the alternating current electric field. Therefore, the pores are equally formed in the same part of the cell. For this reason, DN in the medium
If A is included, DNA can be applied to many cells under the same conditions.
You can import A, etc.

Further, according to the cell electroporation device of the present invention, the first electrode forms an AC electric field for producing a pearl chain phenomenon, and the second electrode forms a DC pulsed electric field for electroporation. It turns out. Here, the second
If the electrode is formed of a plurality of electrode members, it becomes possible to make the crowbar chain phenomenon caused by the first electrode more linear.

〔Example〕

Hereinafter, one embodiment of the present invention will be described with reference to the accompanying drawings.

FIG. 1 shows the main parts of a cell electroporation device according to an embodiment. As shown in the figure, a recess 11 having a substantially square planar shape is formed in the center of the thin substrate 10, first electrodes 3A and 3B are provided on a pair of side walls facing each other, and a second electrode is provided on the other side wall. Two electrodes 6A and 6B are arranged. Electrode 3
AC voltage v1 from an AC power supply is applied to electrodes A and 3B via switch S, and DC pulse voltage V2 from a DC pulse power supply is applied to electrodes 6A and 6B via switch S2. It is now possible to do so.

Then, the medium containing the cells is dripped into the recess 11.

The substrate 10 is made of a transparent material such as quartz glass, and has a light source (not shown) disposed below and a microscope above so that the state of the cells can be observed. The recess 11 has a size of about 1 crI1 on one side, and its depth may be about several inches. In addition, the first electrode 3
A, 3B and second electrodes 6A, 6B are platinum (Pt)
It is formed by etc.

The alternating current voltage (1) applied to the electrodes 3A and 3B has a frequency of about several MHz and has a strength to form an electric field of about 10 to IOOV/1 in the medium. In addition, electrodes 6A, 6
The DC pulse voltage V2 applied to B has a pulse width of 10~
At about 20μSee, 10 to 10'V/am in the medium
It is strong enough to form an electric field of approximately The voltage applied to the cell membrane when the cell membrane is destroyed is constant (1v) regardless of the type of cell, but the frequency of the alternating current electric field and the waveform, length, and size of the high voltage pulse depend on the temperature at the time of the pulse. It goes without saying that the values may be changed as appropriate depending on the salt environment, the size and type of cells, the properties of the medium, etc., and are not limited to the above values.

Next, the operation of the above embodiment will be explained with reference to FIG.

First, the medium 2 containing the cells 1 is dripped into the recess 11 in a state l2 with the switch S 18 turned off. Then, when the switch Sl is turned on, the cells 1 are arranged in a line due to the crowbar chain phenomenon. FIG. 2(a) shows this state, which can be seen by providing a light source below the substrate 10 and observing it from above with a microscope. next,
When the switch S2 is turned ON and a DC pulse is applied in a direction perpendicular to the AC electric field, a small pore 4 is formed in the cell 1 as shown in FIG. 2(b). Then, the DNA in the medium 2 is taken into the cell 1 through the small pore 4 of this cell membrane. Immediately after that, when the switch S2 is turned OFF and the application of the DC pulsed electric field is canceled, the small pores 4 in the cell membrane are gradually repaired, and as a result, the DNA is taken into the cell 1.

In this case, the cells 1 are arranged in a line while facing in a fixed direction by an alternating current electric field, and a direct current pulsed electric field for forming the small pores 4 is applied in a fixed direction. For this reason,
For all cells 1, small pores 4 of approximately the same size can be formed at the same site.

Furthermore, since the position of the formed small pore 4 is different from the arrangement direction of the cells 1, the cells 1 will not fuse with each other during the repair process of the small pore 4.

FIG. 3 shows the main parts of a cell electroporation device that is a modification of the above embodiment. In this example, the second electrodes 6A, 6B for applying the DC pulse voltage (2) are each formed by four electrode members 6At to 6A46B to 6B4. and ■ Electrode members 6A to 6A4 on one side wall are switches S21.
The electrode parts 6B to 6B4 on the other side wall are connected to the DC pulse voltage 2 by the switch S22, but when the switch S21 and the switch S22 are turned OFF (the state shown in the figure), ), each electrode member 6A to 6A, 6
B to 6B4 are electrically isolated from each other.

When performing electroporation according to this modification, first, the medium 2 containing the cells 1 is dripped into the recess 11 of the substrate 10. Then, by turning on the switch Sl and applying an AC voltage 1 to the electrodes 3A and 3B, a pearl chain phenomenon is caused and the cells 1 are arranged. Here, the arrangement direction of the cells 1 is along the direction of the electric lines of force of the alternating current electric field, but in this modification, the second electrodes 6A and 6B are divided into four parts and are electrically separated from each other, so that First electrodes 3A, 3B
The lines of electric force due to the AC voltage V1 between the second electrodes 6A, 6
It is kept substantially straight without being affected by B. Therefore, the position of the small pore 4 formed in the cell membrane of the cell 1 after turning on the switches S 1 and S 2 can be made more constant for all the cells 1.

Note that the first electrodes 3A, 3B and the second electrodes 6A, 6
Various modifications can be made to the shape and positional relationship of B. For example, the electrode 6A may be provided as a transparent electrode on the substrate 10 on the bottom surface of the recess 11, and the electrode 6B may be provided as a transparent electrode on the lid of the four parts 11.

Next, specific examples and comparative examples by the present inventor will be described.

First, a quartz glass plate was prepared in which a concave portion with a side of 1 cm and a depth of 31I1 was formed in the center, and electrodes as shown in FIG. 1 were formed on the four parts using platinum (Pt). An AC voltage source with variable voltage and frequency was connected to one pair of electrodes, and a DC pulse power source with variable voltage and pulse width was connected to the other pair of electrodes.

Furthermore, a white light source was placed below the substrate made of quartz glass, and an objective lens of an optical microscope was placed above the recess. Using the above device, we conducted experiments with sea urchin eggs and human red blood cells.

Example 1 1 ml of a sucrose aqueous solution having approximately the same concentration as seawater was prepared, and approximately 500 live sea urchin eggs were placed therein to serve as a sample solution. Next, this sample solution was dripped onto the four parts of the quartz glass plate to a depth of 1 to 2 degrees, and the electric field strength was 200 V/cm.
A MHz alternating current electric field was applied.

After confirming the occurrence of the pearl chain phenomenon using a microscope,
When pulses with an electric field strength of 400 V/cm and a pulse width of 50 μSee were applied five times at 1 second intervals, perforation was observed in sea urchin eggs. These perforations were formed in approximately the same location and with approximately the same size in more than 98% of sea urchin eggs. Furthermore, even after the pores in the cell membrane were repaired, the sea urchin eggs did not fuse with each other.

Comparative Example 1 Using the same sample as in Example 1, a DC pulse was applied without applying an AC electric field. As a result, about 40% of all sea urchin eggs had perforations. Additionally, the location and size of the perforation were not consistent.

Example 2 1 ml of a sucrose aqueous solution having approximately the same concentration as human blood was prepared, and about 1,000 live red blood cells were added thereto to form a sample solution. Next, apply this sample solution to the concave part of the quartz glass plate.
Drop down to a depth of 2■■, and apply 300 V/
An alternating current electric field of IMHz was applied with a field strength of cm. After confirming the occurrence of the crowbar chain phenomenon using a microscope, 1. O
When pulses with an electric field strength of KV/cm and a pulse width of 5 μSee were applied only 5 times at 1 second intervals, perforation of red blood cells was observed. This perforation was formed at approximately the same site and with approximately the same size in more than 98% of the red blood cells. Furthermore, even after the small pores in the cell membrane were repaired, the red blood cells did not fuse with each other.

Comparative Example 2 Using the same sample as in Example 1, a DC pulse was applied without applying an AC electric field. As a result, about 40% of the red blood cells were perforated. Additionally, the location and size of the perforation were not consistent.

〔Effect of the invention〕

As described in detail above, in the present invention, cells are arranged in a fixed direction by an alternating current electric field, and these cells are perforated by a direct current pulsed electric field in a direction crossing the alternating current electric field. Therefore, the pores are equally formed in the same part of the cell. Therefore, by including DNA or the like in the medium, it is possible to incorporate the DNA or the like into a large number of cells under the same conditions.

In the present invention, small pores can be formed in cells in a medium with good controllability, so that it can be widely applied to industrial applications related to biotechnology.

[Brief explanation of drawings]

FIG. 1 is a perspective view of the main parts of a cell electroporation device according to an embodiment of the present invention, FIG. 2 is a diagram explaining the operation of the embodiment, and FIG. 3 is an embodiment of the embodiment shown in FIG. 1. FIGS. 4 and 5 are perspective views showing essential parts of a modified example of 1. Cells, 2. Medium, 3A, 3B. First electrodes. , 4... Small hole, 6A, 6B... Second electrode, 1
0...Substrate, 11...Recess, ■...AC voltage,
V2...DC pulse l voltage. Patent applicant Hamamatsu Photonics Co., Ltd. Representative Patent Attorney
Hasegawa Yoshi Itsuki Measuring Detective t,m

Claims (1)

[Claims] 1. In a cell electroporation method in which small pores are formed in the cell membrane of cells contained in a medium, an alternating current electric field is applied to the medium, and the cells are moved in the direction of the electric field of the alternating electric field. Cell electricity comprising: a first step of arranging the cells; and a second step of applying a DC pulsed electric field in a direction crossing the electric field direction of the AC electric field to form small pores in the cell membrane of the cells. perforation method. 2. The cell electroporation method according to claim 1, wherein the medium contains in advance a substance to be taken into the cells. 3. a base having a recess formed therein for containing a medium containing cells; a pair of first electrodes disposed on the base so as to be able to apply an electric field to the medium placed in the recess; the first of
a pair of second electrodes arranged on the base body so as to be able to apply an electric field to the medium in a direction crossing the direction of the electric field generated by the electrodes; and an AC power source that supplies an AC voltage to the first electrode. A cell electroporation device comprising: means for supplying a DC pulse voltage to the second electrode after supplying the AC voltage to the first electrode. 4. The second electrode is composed of a plurality of electrode members,
4. The cell electroporation apparatus according to claim 3, wherein the plurality of electrode members are electrically isolated from each other until immediately before the application of the DC pulse voltage.