JPH02131585A - Method and device for electrically boring hole - Google Patents

Abstract

PURPOSE:To selectively and readily take a taking-in material in a specific site of a cell by positioning a taking-in part of cell on either one side of direction of line of electric force, heating a cell part on other side and applying direct current pulse electric field in providing small hole for taking-in material in the cell by an electrically boring method. CONSTITUTION:A medium liquid 17 containing a cell 16 is put in a recessed part 11 formed in the central part of a substrate 11 and DNA taking-in part of the cell 16 is positioned on one side in the direction of line of electric force by a method rotating and moving the substrate 11 while observing using an optical microscope 3. Then a part of cell 16 on other side in the direction of line of electric force is irradiated with laser beam L1 from laser light source 4 and selectively heated. At the same time, electricity is sent to electrodes 13a and 13b from a direct current pulse electric source 2 and direct current pulse electric field is applied to the medium liquid 17 to form small holes. Thereby DNA, etc., is taken in from the small holes for a long period, since repair of small holes in unheated position of cell 16 is slow.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a cell electroporation method in which DNA and the like are taken into cells by applying a high-voltage pulsed electric field to a medium containing cells and forming small pores in the cell membrane. This invention relates to a cell electroporation device used for this purpose.

[Conventional technology]

With the progress of biotechnology, it has become important to incorporate substances such as DNA into cells. In order to carry out such operations, it is necessary to form small holes in the cell membrane, and the first technique for this is a perforation method using laser light. Some methods involve heating a specific part of a cell by irradiating it with a laser beam to form small pores in the cell membrane. According to this method, DNA is taken into the cell through this small pore, but since the small pore is formed by heating in this method, the cell membrane is easily damaged, and the D
Small pores will remain even after NA uptake.

On the other hand, according to the electroporation method, which is the second conventional technique, small pores are formed in the cell membrane by applying a DC pulsed electric field to a medium containing cells, and DNA is transferred through the small pores. etc. will be imported. According to this electroporation method, the cell membrane is not particularly damaged, and the small hole is repaired when the application of the DC pulsed electric field is stopped.

[Problem to be solved by the invention]

However, in the case of electroporation, in principle, small pores are formed in the cell membranes of all cells in the DC pulsed electric field, so it is not possible to incorporate DNA into only a specific cell among a large number of cells.

In addition, since these small holes are formed equally at one end and the other end in the direction of the electric field lines of the DC pulsed electric field, DNA can be taken into only a specific part of one cell, for example, the nucleus of the cell or its vicinity. That is difficult.

Therefore, the present invention provides a cell electroporation method that allows the incorporation of a substance such as DNA into only a specific cell among a plurality of cells in a medium or a specific site of a single cell, and a cell electroporation method for this purpose. The purpose is to provide a device for use.

[Means for solving intelligence problems]

In the cell electroporation method according to the present invention, a small pore is formed by applying a direct current pulsed electric field to the cell membrane of a cell contained in a medium, and through this small hole, the cell membrane of the cell contained in the medium is In the cell electroporation method, in which a target material such as DNA is taken into the uptake area of the cell, the uptake area of the cell (e.g. near the nucleus) is placed on one side in the direction of the electric field lines of the DC pulsed electric field.
and a second step of selectively heating the cell portion on the other side in the direction of the electric lines of force with a laser beam or the like and applying a DC pulsed electric field. .

In addition, in the cell electroporation method according to the present invention, when a plurality of cells are included in the medium, a predetermined cell among the plurality of cells is transferred through a small hole formed by applying a DC pulsed electric field. When taking the target material, cells other than the above-mentioned predetermined cells are selectively heated with a laser beam or the like, and
The method may also include a step of applying a DC pulsed electric field.

Furthermore, the cell electroporation device according to the present invention includes a base body in which a recess is formed for containing a medium containing cells, and a pair of electrodes for applying an electric field to the medium are provided in the recess;
a power source that applies a DC pulse voltage to the pair of electrodes;
It is characterized by comprising a microscope for observing cells in a medium placed in a recess in a base, and a laser light source for heating cells in a medium placed in a recess in a base.

[Effect]

According to the configuration of the present invention, only a specific part of a single cell in a medium or a part of a plurality of cells is selectively heated, and under this condition, electroporation by a DC pulsed electric field is performed. It will be done. Here, since the electroporated pores in the cell membrane are repaired quickly under heating conditions, the time the pores remain open is relatively short. On the other hand, the small pores in the cell membrane of specific areas or specific cells that are not heated will be repaired slowly, and the time that the pores remain open will be relatively long. DNA etc. will be taken in. Therefore, it becomes possible to selectively incorporate DNA into a specific site of a cell or a specific cell.

〔Example〕

Embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is a basic configuration diagram of an apparatus to which an embodiment of the cell electroporation method according to the present invention is applied, and FIG.
FIG. As shown in FIG. 2, the base body 1 has a substrate 11 made of, for example, quartz glass, and a recess 12 having a quadrilateral planar shape is formed in the center of the substrate 11. Recess 12
A pair of electrodes 13a and 13b are formed of, for example, platinum (Pi) on opposing side walls of the
a and 14b are connected. Recessed portion 12 of this substrate 11
A predetermined amount of the medium 17 containing the cells 16 is put into the container, and the container is set as shown in FIG.

That is, as shown in FIG.
b is connected to the DC pulse power source 2, which causes the medium 1
A DC pulse electric field is applied to 7. The state of the cells 16 is observed using an optical microscope 3, and the cells 16 in the medium 17 are irradiated with a laser beam L1 from a laser light source 4 via the optical microscope 3.

On the other hand, a white light source 6 is provided below the substrate 1 via a relay lens 5, and the light from this source passes through the cells 16 and is magnified by the optical microscope 3, and reaches the observer 7 as observation light L2. ing. Note that M in the figure is a total reflection mirror, and BS is a beam splitter.

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

In FIG. 3, cells 16 contained in a medium (not shown)
is assumed to have a nucleus 16a. Now, when a DC pulsed electric field is applied between the electrodes 13a and 13b in the state shown in FIG. Ru. This small hole becomes open by applying a DC pulsed electric field, and repairs itself when the DC pulsed electric field is removed.

Incidentally, the rate of formation and repair of these small pores varies depending on the temperature of the part; when the temperature is high, both the formation and repair are rapid, and when the temperature is low, the formation and repair are both slow. Therefore, when a laser beam is irradiated only to the part indicated by the symbol LA in Fig. 3 and a DC pulse electric field is applied while heating this part, the small pores of the cell part Pt in the part LA heated by the laser beam are The total time that the pores are in the open state is relatively short, and the total time that the small pores in the other unheated cell portion P2 are in the open state is relatively long.

As a result, more DNA in the medium 17 is taken in through the small pores of the unheated cell portion P2.

Specific procedures for incorporating DNA include the following.

First, since what is electroporated is the cell membrane at the end in the direction of the lines of electric force of the DC pulsed electric field, the uptake part where DNA is to be taken in is positioned in the direction of the lines of electric force of the DC pulsed electric field. As a specific method for this, for example, the substrate 1 may be rotated and moved while being observed with an optical microscope 3. Alternatively, for example, by applying a high frequency electric field, a well-known crowbar chain phenomenon may be caused to direct the cells 16 in a specific direction.Next, the identification of a specific cell or a single cell among multiple cells Heating of the site is performed by laser light irradiation, but it is desirable to stain the cells 16 prior to this laser light irradiation. As the dye, a low luminescent dye that is less toxic to the cells 16 is used.

In this way, by using a pulse laser or a CW laser, a specific portion can be selectively heated by irradiating the laser beam. The selective laser beam irradiation is performed by scanning the laser beam while checking the position and orientation of the cells 16 using the optical microscope 3. on the other hand,
By staining only the part of a single cell to be heated or a specific cell to be heated among multiple cells, selective heating can be performed even if the entire area is irradiated with laser light.

Next, under the heating conditions described above, a DC pulsed electric field is applied to perform electroporation. This DC pulse electric field is 1 to several μs
This is repeated several times with a pulse width of ec and a pulse interval of 1 to several seconds. Note that the application timing of the DC pulsed electric field and the irradiation timing of the laser beam may be any timing as long as it can selectively heat the cell membrane during the formation or repair process. When electroporation is performed as described above, the DNA contained in the medium 17 is selectively taken into the cells 16.

FIG. 4 specifically shows some examples of DNA uptake. In the figure, symbol E indicates the direction of electric lines of force due to the DC pulsed electric field, and symbol LA indicates a portion heated by laser beam irradiation.

Further, the hatched portion in the figure is a portion where a relatively open large hole is formed. According to the example in the figure (a), it is possible to controllably (DNA) only be taken into the vicinity of the nucleus 16a. Only in D
NA can be taken in. According to the example shown in FIG. 4(C), DNA can be selectively taken into only two cells out of seven cells. Also, according to the example in figure (d),
DNA can be selectively taken up by only one cell among four divided cells.

Next, a specific example by the present inventor will be described.

First, a quartz glass plate was prepared in which a concave portion having a side of 1 clI1 and a depth of 3 mm was formed in the center, and an electrode pair as shown in FIG. 2 was formed in this concave portion using platinum (Pi). A DC pulse power source with variable voltage and pulse width and a pulse interval of 1 second was connected to the electrode pair. 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. Furthermore, argon (A
r) The laser device was set up to enable laser beam irradiation. Using the above device, we conducted experiments using sea urchin eggs and human red blood cells stained with carotene dyes.

Example 1 1 ml of a sucrose aqueous solution having approximately the same concentration as seawater was prepared, and approximately 50 live sea urchin eggs were placed therein to serve as a sample solution.

Next, apply this sample solution to the recess of the quartz glass plate by 1 to 2 mm.
The sea urchin eggs were dropped to a depth of 100 cm and checked under a microscope to select the sea urchin eggs to be treated. Next, A with a spot diameter of 5 μm
The r laser beam is scanned and irradiated on only one side of the selected cell, and a DC pulsed electric field with a pulse width of 5 μsec and a pulse interval of 1.0 sec is applied at 1.5 KV/co+.
It was applied 10 times with a strength of . As a result, five times as much DNA in the sucrose aqueous solution was incorporated into the unheated sea urchin egg cells as compared to the heated part.

Comparative Example 1 In Example 1, a DNA uptake experiment was conducted under exactly the same conditions except for the laser beam irradiation. As a result, the amount of DNA taken in was approximately the same in the direction of the electric lines of force of the DC pulsed electric field.

Example 2 1 ml of a sucrose aqueous solution having approximately the same concentration as human blood was prepared, and approximately 50 live red blood cells were placed therein to form a sample solution. Next, apply this sample solution to the concave part of the quartz glass plate for 1 to 2 I
The solution was dropped to a depth of II+ and confirmed under a microscope to select only 6 red blood cells to be treated. Next, an Ar laser beam with a spot diameter of 5 μm was scanned and irradiated onto only 4 of the 6 selected cells, and the pulse width was 1, 5 μm.
A DC pulsed electric field of μsec and a pulse interval of 1.0 sec was applied 10 times at an intensity of 1.5 V/cm. As a result, five times as much DNA in the sucrose aqueous solution was incorporated into two unheated red blood cells as compared to four heated red blood cells.

Comparative Example 2 In Example 1, a DNA uptake experiment was conducted under exactly the same conditions except for the laser beam irradiation. As a result, the amount of DNA uptake was approximately the same in the six red blood cells.

〔Effect of the invention〕

As explained above in detail, in the present invention, only a specific part of a single cell in a medium or a specific cell among a plurality of cells is selectively heated, and under this condition, a DC pulsed electric field is applied. Since electroporation is performed, specific parts of cells that have not been heated or small pores in the cell membrane of specific cells will be repaired slowly, and DNA etc. will be taken in from here over a long period of time. Therefore, it becomes possible to selectively incorporate DNA into a specific site of a cell or a specific cell.

[Brief explanation of the drawing]

FIG. 1 is a basic configuration diagram of a cell electroporation device according to an embodiment of the present invention, FIG. 2 is a perspective view of the base shown in FIG. 1, and FIG.
The figure is a diagram for explaining the effect of the embodiment, and FIG. 4 is a diagram for explaining an example of DNA uptake according to the embodiment. DESCRIPTION OF SYMBOLS 1... Base body, 12... Recessed part, 13a. 13b...
Electrode, 2... DC pulse power supply, 3... Optical microscope,
4... Laser light source, 5... Relay lens, 6...
White light source, 7... Observer patent applicant Hamamatsu Photonics Co., Ltd. Representative patent attorney
Yoshiki Hasegawa Figure 1: #+ perspective view of the toad body Figure 2: The original structure of the 1st part that breathes into the actual situation Figure 1: Action and explanatory diagram of the actual example Figure 3: r

Claims (1)

[Scope of Claims] 1. A small pore is formed by applying a direct current pulsed electric field to the cell membrane of the cell contained in the medium, and the material previously contained in the medium is passed through the small pore. In a cell electroporation method in which a substance is taken into an uptake part in the cell, a first step of locating the uptake part of the cell on one side in the direction of the lines of electric force of the DC pulsed electric field; A cell electroporation method comprising a second step of selectively heating the cell portion on the other side in the linear direction and applying the DC pulsed electric field. 2. Cell electroporation according to claim 1, wherein the cells are dyed in advance, and the second step includes selectively irradiating and heating the cell portion on the other side in the direction of the lines of electric force with a laser beam. Law. 3. The cell according to claim 1, wherein the cell portion on the other side of the electric force line direction is dyed in advance, and the second step includes heating the dyed portion by irradiating the entire cell with a laser beam. Electroporation. 4. A small pore is formed by applying a DC pulse electric field to the cell membrane of a plurality of cells contained in the medium, and a predetermined cell among the plurality of cells is introduced into the medium through this small pore. In the cell electroporation method in which pre-incorporated substances are taken, cells other than the predetermined cells are selectively heated, and
A cell electroporation method comprising the step of applying the DC pulsed electric field. 5. The cell electroporation method according to claim 4, wherein the selective heating is performed by selectively irradiating cells other than the predetermined cells with laser light. 6. The cell electroporation method according to claim 4, wherein the selective heating is performed by selectively staining cells other than the predetermined cells and irradiating them with laser light. 7. A base having a recess formed therein for containing a medium containing cells, and a pair of electrodes provided in the recess for applying an electric field to the medium, and applying a DC pulse voltage to the pair of electrodes. a microscope for observing the cells in the medium placed in the recess of the base; and a laser light source for heating the cells in the medium placed in the recess of the base. A cell electroporation device comprising: