JP2016106526A - Method for introducing material and device therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for introducing material with which introduction material is introduced into a target cell while suppressing electrical damage with the use of electroporation.SOLUTION: The method for introducing material for introducing an introduction material into a target cell comprises a burst pulse application step for applying a burst pulse in which an asymmetrical reference pulse constituted by keeping a level of a fixed potential value for a predetermined time period during positive and negative change of polarity is repeated in the same period, to a target cell immersed in solution containing the introduction material, and a high-voltage pulse application step for applying a high voltage pulse that has voltage higher than that of the reference pulse constituting the burst pulse and is shorter in time, to the target cell for one ON time after the application by the burst pulse application step. The method introduces the introduction material to the target cell while suppressing electrical damage.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for introducing a substance to be introduced into a target cell, and particularly relates to a method for introducing a substance to be introduced using an electric field.

  In recent years, the need for a technique for introducing a substance to be introduced into living cells has increased. For example, the introduction of test substances into fertilized eggs such as medaka for the purpose of evaluating the biological effects of chemical substances, and the introduction of genes into living cells of animals as gene therapy experiments have been There are many fields. Conventionally, a microinjection method is known as a technique for introducing such an introduced substance. The microinjection method is a method of introducing a substance to be introduced by inserting a glass needle into a target cell. However, since a physical force is applied in the operation, a load is applied to the target cell or the target cell is destroyed. Cases can arise.

  As a method for solving such a problem, it has been proposed to introduce a target substance into a target cell by using an electric force. For example, there is a method of introducing an introduced substance into a target cell by applying an electric pulse between electrodes to form an electric field and attracting electrophoresis of the introduced substance by the electric field (see Patent Document 1). Further, as an electroporation method for introducing a drug into a cell (electroporation), a method of introducing a drug into a cell by applying an electric field pulse such as a square wave pulse or an exponential wave pulse (see Patent Document 2) And a method of producing a genetically modified plant by introducing a polynucleotide into a plant cell (see Patent Document 3). In addition, as an in vivo electroporation therapy (EPT method), a method of killing cells using a low voltage long pulse length is also disclosed (see Patent Document 4).

  Furthermore, the present inventors have also attempted to introduce the target substance by electroporation in which an electric pulse is applied mainly to fertilized eggs of medaka. For example, investigation of the relationship between molecular weight and introduction efficiency in substance introduction into medaka eggs by electric pulse (Non-Patent Document 1), and use of an electric pulse with a pulse width of 50 to 300 ns for the applied electric pulse (non- Patent Document 2), using an electric pulse with a pulse width of 15 ns and a voltage value of 6 kV (Non-Patent Document 3), and using a high electric field rectangular wave pulse (Non-Patent Documents 4 and 5) are disclosed. In addition, a method of applying two types of electric pulses in succession is also disclosed (Non-Patent Documents 6 and 7). Among them, the application of a high-voltage rectangular wave pulse having a pulse width of 100 nanoseconds, The structure which combines the application of the low voltage decay wave pulse of this is also disclosed (nonpatent literature 8).

JP 2004-041023 A Special table 2001-517998 Special table Hei 11-511974 public information Special table 2000-503586

Akemi Yamaguchi, Yasutaka Imamura, Atsushi Kono, Nobuaki Tominaga, “Introduction of Substances into Medaka Eggs by High Electric Field Pulses; Examination of Molecular Weight and Introduction Efficiency” Proceedings of the Kyushu Branch Joint Conference on Electrical Engineering (CD-ROM), 2011 September 16, Vol.64th Page.ROMBΜNNO.04-1P-16 NAKAMITSΜ S., KANG DK, YAMANAKA M., HOSSEINIS.HR, SHIRAISHI E., SAKUGAWA T., KATSΜKI S., AKIYAMA H., KONO S., TOMINAGA N. Embryogenesis ”IEEJ Pulse Power Study Group, 2009.October 23, Vol.PPT-09 No.102-107 Page.27-31 Takashi Tanabe, Akemi Yamaguchi, Nobuaki Tominaga, Atsushi Kawano “Basic Research on Introduction of Highly Efficient Substances into Medaka Eggs Using Nanosecond Pulse Power” IEEJ Pulse Power Study Group Material, January 7, 2010, Vol.PPT -10 No.1-10 Page.1-5 "Nakajima Atsushi, Yamaguchi Akemi, Tanabe Takashi, Kono Atsushi, Tomonaga Nobuaki" Introduction of Substances into Medaka Eggs Using Ultra-short High Electric Field Pulses "Japan Society for Agricultural Chemistry, West Japan Branch Meeting and Symposium Proceedings, September 17, 2010 Sun, Vol.2010 Page.38 Akemi Yamaguchi, Kaoru Kono, Takashi Tabuchi, Nobuaki Tominaga “Introduction of Substances into Fertilized Eggs of Different High-Field Square-Wave Pulses” Proceedings of the Annual Conference of the Institute of Electrical Engineers of Japan, March 5, 2011, Vol.2011 No.1 Page.239 Satoshi Kawano, Akemi Yamaguchi, Nobuaki Tominaga "Consecutive application of two kinds of voltage pulses for the purpose of increasing the amount of chemicals introduced into the medaka egg" Proceedings of the IEEJ Fundamentals, Materials and Common Section Conference (CD- ROM), September 21, 2011, Vol.2011 Page.ROMBΜNNO.B-7 Satoshi Kawano, Akemi Yamaguchi, Takashi Tabuchi, Nobuaki Tominaga "Introduction of Substances into Fertilized Eggs of Medaka by Continuous Application of Two Different Pulse Voltages" Proceedings of the Annual Conference of the Institute of Electrical Engineers of Japan, March 5, 2011, Vol.2011 No .1 Page.240 Takahiro Kubo, Akemi Yamaguchi, Yasutaka Imamura, Nobuaki Tominaga, Satoshi Kawano "Study of parameters in substance introduction experiment to medaka fertilized eggs using pulse power" IEEJ Pulse Power Study Group, March 8, 2012 , Vol.PPT-12 No.1-4.6-8 Page.5-8

  However, in the conventional method for introducing a substance using an electric force, various voltage values and application methods that can be optimized when applying a voltage have been sought. There is a problem that the target cell itself is damaged, and the target cell (for example, a living cell such as a fertilized egg) itself is destroyed depending on the voltage. Further, as shown in Non-Patent Document 3, a configuration in which two different voltage applications are combined has been proposed, but a remarkable effect on introducing a substance into a target cell has not been confirmed. Furthermore, introduction of a gene such as a plasmid into a target cell has not yet been reported.

  In order to solve the above problems, an object of the present invention is to provide a substance introduction method for introducing an introduction substance into a target cell while suppressing electrical damage using an electroporation method.

  The substance introduction method disclosed in the present application is a substance introduction method in which an introduction substance is introduced into a target cell (eg, a living cell such as a fertilized egg). A constant potential value level is set for a predetermined time while the polarity changes positively or negatively. A burst pulse that is applied to a target cell (for example, a living cell such as a fertilized egg) immersed in a solution containing the introduced substance, a burst pulse in which the maintained asymmetric reference pulse is repeated in the same cycle. An application step, and a high-voltage pulse application step of applying a high-voltage pulse that is higher in voltage and shorter than the burst pulse to the target cell for one on-time after the application in the burst pulse application step. .

  As described above, the substance introduction method disclosed in the present application includes a burst pulse in which an asymmetric reference pulse that is maintained at a constant potential level for a predetermined time while polarity changes positively and negatively is repeated in the same cycle. After applying to the target cell immersed in the solution to be applied, a high voltage pulse having a higher voltage and a shorter time than the reference pulse constituting the burst pulse is applied to the target cell for one on time. Therefore, by a burst pulse (asymmetric burst pulse) in which an asymmetric reference pulse is repeated at the same period, an outer membrane (eg, egg membrane or cell membrane) of a target cell (eg, a target organism of a fertilized egg) In addition, since either one of the positive and negative electrodes is not continuously charged with a biased polarity, it is possible to suppress the electrical damage while at the same time introducing the conductive material immersed in the solution. When the substance is moved in either the positive or negative direction, a situation where the introduced substance is easily introduced is formed. Under this situation, a high voltage pulse is applied for a short time, so that the target cell is introduced. Damage to tissues in the outer membrane by forming temporary holes in the outer membrane (eg, egg membrane, cell membrane, etc.) of the fertilized egg (eg, the target organism) and minimizing the effects The introduction substance can be introduced into the target cell (for example, the target organism of the fertilized egg).

  In the substance introduction method disclosed in the present application, the reference pulse constituting the burst pulse has a peak value that differs between positive and negative polarities as necessary. Thus, in the substance introduction method disclosed in the present application, the intensity of the pulse applied to the target cell is alternately changed over time due to the difference in the peak value between the positive and negative polarity, so that the outer membrane is charged. While suppressing electrical damage by adjusting the amount of charge, there is a situation where the introduced substance is more easily introduced by moving the introduced substance immersed in the solution in either the positive or negative direction. In this situation, by applying a high voltage pulse for a short time, a larger amount of introduced substance can be introduced into the target cell at once without damaging the target cell.

  In the substance introduction method disclosed in the present application, the reference pulse constituting the burst pulse has a duty ratio different between positive and negative polarities as necessary. As described above, the substance introduction method disclosed in the present application is charged to the outer membrane because the application timing of the pulse applied to the target cell alternately changes with time due to the difference in duty ratio between positive and negative polarity. In the situation where the introduced material is more easily introduced by moving the introduced material immersed in the solution in either direction of the positive or negative electrode while suppressing the electrical damage by adjusting the charge amount In this situation, by applying a high voltage pulse for a short time, a larger amount of the introduced substance can be introduced into the target cell at once without damaging the target cell.

  In the substance introduction method disclosed in the present application, the burst pulse is configured by sequentially attenuating the applied charge amount of each reference pulse as necessary. As described above, in the substance introduction method disclosed in the present application, the intensity of the burst pulse becomes smaller than the reference pulse at the initial stage of the burst pulse application immediately before the subsequent high voltage pulse is applied. The difference between the peak value of the voltage pulse and the burst pulse is increased, and a situation where the introduced substance is more easily accepted for the target cell is formed, and the high voltage pulse is applied under the situation, More introduction substances can be introduced into the target cells without damaging the target cells.

  In the substance introduction method disclosed in the present application, if necessary, the reference pulse constituting the burst pulse has an on time in the order of microseconds and an applied voltage in the order of several tens to several hundreds V, and the high voltage pulse Have an on-time of nanosecond order and an applied voltage of kV order. Thus, the substance introduction method disclosed in the present application has an on time in the order of microseconds and an applied voltage in the order of several tens to several hundreds V, and the high voltage pulse has an on time in the order of nanoseconds and kV. Since the applied voltage has an order, the optimum combination of the burst pulse and the high-voltage pulse allows the introduced substance that is efficiently immersed in the solution by the burst pulse to move in either the positive or negative direction. A situation in which the cells are likely to receive the introduction substance is formed, and by applying the subsequent high voltage pulse, more introduction substance can be introduced into the target cell without damaging the target cell. .

  In the substance introduction method disclosed in the present application, the high voltage pulse is applied with the same polarity as that of the polarity side with a small amount of applied charge among the positive and negative reference pulses constituting the burst pulse, as necessary. is there. Thus, since the high voltage pulse is applied with a polarity different from the polarity of the charge charged in the target cell by the burst pulse, the charge having the same polarity as the charge charged in the target cell by the burst pulse. As a result, it is possible to suppress excessive damage to the target cell due to the superposition of the high-voltage pulse, and by introducing the high-voltage pulse, more introduction without damaging the target cell. The substance can be introduced into the target cell.

  In the substance introduction method disclosed in the present application, the applied charge amount of each positive and negative reference pulse constituting the burst pulse has a ratio of 10: 1 to 1:10 between the positive and negative as necessary. Thus, in the substance introduction method disclosed in the present application, the applied charge amount of each positive and negative reference pulse constituting the burst pulse has a ratio of 10: 1 to 1:10 between each positive and negative. While controlling electrical charge by controlling the amount of electric charge charged to the cell, the introduced cell immersed in the solution moves in either the positive or negative direction, so that the target cell becomes the introduced material. An optimal situation is formed in which a large amount of introduced substance can be introduced into the target cell without damaging the target cell by the subsequent application of the high voltage pulse.

  The substance introduction method disclosed in the present application is, as necessary, maintained at a constant potential value level for a predetermined time while the polarity changes positively or negatively after the high voltage pulse is applied by the high voltage pulse applying step. And a subsequent burst pulse applying step of applying a burst pulse in which an asymmetric reference pulse is repeated in the same cycle to the target cell. In this way, a burst pulse that is weaker than the high voltage pulse is further applied to the target cell into which the introduction substance has been introduced by the application of the high voltage pulse, in a subsequent burst pulse application step. The introduction of the introduced substance is promoted by further electrical stimulation to the target cell, and the introduction of the introduced substance to the target cell can be further ensured.

  In the substance introduction method disclosed in the present application, if necessary, the peak value of the first reference pulse constituting the burst pulse applied in the subsequent burst pulse applying step is applied in the burst pulse applying step corresponding to the previous step. The peak value of the last reference pulse constituting the burst pulse is equal to or less than the peak value. As described above, since the target cell into which the introduction substance has been introduced is given a more gentle electrical stimulation by the subsequent burst pulse application step, the introduction of the introduction substance into the target cell is stable. Thus, the introduction substance can be more reliably introduced into the target cell.

  The substance introduction apparatus disclosed in the present application is a substance introduction apparatus that introduces an introduction substance into a target cell, a storage means that contains the solution containing the introduction substance and the target cell, and is disposed around the storage means. A pair of counter electrodes, a DC power source that supplies a DC current that generates a voltage to be applied between the counter electrodes, and a constant while the polarity changes positively or negatively based on the DC current supplied from the DC power source. A burst pulse in which an asymmetric reference pulse having a potential level maintained for a predetermined time is repeated in the same period, and a high voltage pulse having a higher voltage and a shorter time than the reference pulse constituting the burst pulse is applied to the target cell. And a pulse control means for controlling the application.

  As described above, the storage means for storing the solution containing the introduced substance and the target cell, the pair of counter electrodes arranged around the storage means, and the direct current that generates a voltage to be applied between the counter electrodes A DC power source that supplies current and an asymmetric reference pulse that maintains a constant potential value level for a predetermined time while polarity changes positively and negatively based on the DC current supplied from the DC power source are repeated in the same cycle. Asymmetric reference pulse comprising: a burst pulse; and pulse control means for performing a control to apply a high voltage pulse having a higher voltage and a shorter time than the reference pulse constituting the burst pulse to the target cell. Since a pulse with a polarity biased to either the positive or negative electrode is applied to the target cell by a burst pulse repeated at the same cycle, While the electrical damage is suppressed by adjusting the amount of charge charged to the outer membrane of the target cell due to the bias, the introduced substance immersed in the solution moves in either the positive or negative direction Thus, an optimal situation is formed in which the target cell can easily receive the introduced substance, and a large amount of the introduced substance is introduced into the target cell without damaging the target cell by the subsequent application of the high voltage pulse. Can do.

  In the substance introduction device disclosed in the present application, as necessary, at least one of the counter electrodes includes a pin-shaped or rail-shaped protrusion on the side facing the other electrode. As described above, in the substance introduction device disclosed in the present application, since at least one of the counter electrodes includes a pin-shaped or rail-shaped protrusion on the side facing the other electrode, Concentration (locality) will increase, concentration of the introduced substance immersed in the solution will increase, and the target cell will be damaged by adjusting the amount of charge charged to the outer membrane of the target cell It is possible to introduce a large amount of the introduced substance into the target cell at once.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic of the substance introduction apparatus which concerns on the 1st Embodiment of this invention, and the flowchart of a substance introduction method are shown. 2 shows an example waveform of an asymmetric burst pulse according to the first embodiment of the present invention. The waveform example of the asymmetrical burst pulse and high voltage pulse which concerns on the 1st Embodiment of this invention, and the circuit structural example of the substance introduction apparatus which concerns on the 1st Embodiment of this invention are shown. Explanatory drawing of the protrusion of the substance introduction apparatus which concerns on the 2nd Embodiment of this invention is shown. The flowchart of the substance introduction method which concerns on the 3rd and 4th embodiment of this invention, and the waveform example of an asymmetrical burst pulse and a high voltage pulse are shown. The experimental result of substance introduction using the substance introduction device concerning the present invention is shown. The introduction result of actinomycin D using the substance introduction apparatus according to the present invention and the occurrence abnormality rate of each chemical substance for substance introduction are shown. The visible photograph and fluorescence micrograph which were image | photographed with respect to the fertilized egg after 1 day progress and 2 days progress after introduction | transduction of the GFP vector by the substance introduction | transduction method based on this invention are shown. The visible photograph and fluorescence micrograph which were image | photographed with respect to the fertilized egg 1 day after the introduction | transduction of the GFP vector by the substance introduction | transduction method based on this invention are shown. It is a GFP vector introduction | transduction experiment result by the substance introduction | transduction method based on this invention, Comprising: The ratio of the fertilized egg by which the fluorescence by GFP was confirmed on the 3rd day after introduction | transduction experiment is shown. The introduction result of actinomycin D performed on the egg on the day of fertilization and the egg on the day after fertilization by the substance introduction method according to the present invention is shown. The introduction experiment result of warfarin sodium by the substance introduction method concerning the present invention is shown. 1 shows a visible photograph taken on a fertilized egg after introduction of loxoprofen sodium dihydrate by the substance introduction method according to the present invention.

(First embodiment)
As shown in FIG. 1 (a), the substance introduction device according to the present embodiment is arranged around a containing means 21 for containing a target cell 100 and a solution 120 containing the introduced substance 110, and around the containing means 21. A pair of counter electrodes 22 and 23 and a voltage (a burst pulse and a high voltage pulse described later) applied between the counter electrode 22 and the counter electrode 23 (for example, a burst pulse generator 41 and a high voltage described later). Based on the DC power supply 3 that supplies the DC current generated by the pulse generator 42) and the DC current supplied from the DC power supply 3, a constant potential value level is maintained for a predetermined time while the polarity changes between positive and negative. A burst pulse in which the asymmetric reference pulse is repeated at the same period, and a high voltage pulse having a higher voltage and shorter time than the burst pulse are applied to the target cell 100. A configuration and a pulse control unit 4 for controlling the.

  The target cell 100 is not limited to a fertilized egg of an organism, and various cells, small animals, plants and the like can be mentioned. Examples of the introduced substance 110 include various inhibitors that inhibit protein synthesis such as cycloheximide (CX) and actinomycin D (AD), various proteins, genes (DNA and RNA), and the like. In addition, a cell labeling reagent (fluorescent labeling reagent for cell staining, oxidative stress labeling reagent, etc.) can also be introduced into the target cell 100 for pharmaceutical use. Further, for example, it can be used for evaluating the influence of a chemical substance (for example, bisphenol A, estradiol, etc.) on a living body. The solution 120 is not particularly limited as long as it is a medium that dissolves the introduction substance 110, and may be in the form of, for example, an aqueous solution, a suspension, or a gel.

  The container 21 can be a plastic container. The counter electrode 22 and the counter electrode 23 are not particularly limited as long as they are generally used electrode materials, and examples thereof include stainless steel, duralumin, copper, and platinum.

  The DC power supply 3 charges a burst pulse generator 41 and a high voltage pulse generator 42, which will be described later, as a pulse power supply that supplies a burst pulse and a high voltage pulse to the counter electrode. That is, the DC power source 3 includes, for example, a burst pulse power source 31 for generating a burst pulse and a high voltage pulse power source 32 for generating a high voltage pulse, as shown in FIG. Composed.

  The pulse control means 4 generates the burst pulse or the high voltage pulse between the counter electrode 22 and the counter electrode 23 by controlling the ON time, the duty ratio, and / or the polarity of the DC voltage of the DC power supply 3. . For example, as shown in FIG. 1A, the pulse control means 4 includes a burst pulse generating unit 41 that generates the burst pulse, a high voltage pulse generating unit 42 that generates the high voltage pulse, and the burst pulse. And timing of applying the high voltage pulse (amount of charge applied to the positive and negative pulses (including pulse width), the number of pulses, and the output interval between the burst pulse and the high voltage pulse) And a timing adjustment unit 43 that adjusts the output and an output control unit 44 that outputs the burst pulse and the high voltage pulse between the counter electrode 22 and the counter electrode 23.

  A substance introduction method using the substance introduction apparatus will be described below with reference to the flowchart of FIG.

  First, the target cell 100 is immersed in the solution 120 containing the introduction substance 110 inside the housing means 21. The target cell 100 immersed in the solution 120 containing the introduced substance 110 has an asymmetric reference pulse in which the zero level is maintained as a constant potential value level for a predetermined time while the polarity changes positively and negatively in the same period. A burst pulse (also referred to as an asymmetric burst pulse) is applied (S1: burst pulse application step). As described above, since the asymmetric reference pulse is repeated at the same period, the target cell 100 is not continuously charged with a charge having a biased polarity on either the positive or negative side. On the other hand, the introduction material immersed in the solution is moved in either direction of the positive and negative electrodes while suppressing the electrical damage to the outer membrane of the film, thereby forming a situation where the introduction material is easily introduced. It will be.

  In the asymmetric burst pulse according to this embodiment, for example, as shown in FIG. 2A, a positive reference pulse (A in the figure) and a negative reference pulse (B in the figure) are different. It can be configured to have a peak value.

  As the peak value, for example, the positive side voltage (Vp) (peak value) can be set to 20V, and the negative side voltage (Vn) (peak value) can be set to -10V. The number (n) of asymmetric burst pulse periods according to the present embodiment can be 1 to 400, but is preferably 100 to 200 from the viewpoint of suppressing electrical damage to the target cell 100. For example, it can be set to 100.

  With such a configuration, the intensity of the pulse applied to the target cell 100 alternately changes with time due to the difference in the peak value between the positive and negative polarity, so that a charge with a biased polarity is applied to either the positive or negative electrode. Since it is not continuously charged, while the electrical damage is suppressed, the introduced substance immersed in the solution is further moved in either the positive or negative direction, thereby introducing the introduced substance. 110 is more easily introduced, and a high voltage pulse is applied for a short time in this situation, so that a larger amount of the introduced substance 110 can be rapidly applied to the target cell without damaging the target cell 100. Can be introduced.

  As described above, the asymmetric burst pulse according to the present embodiment can be configured such that the reference pulse constituting the burst pulse has different peak values on the positive and negative sides, as shown in FIG. In addition, the duty ratio in the positive and negative pulses may be different.

  The on-time and off-time can be several microseconds to several seconds. Regarding the on-time, for example, the on-time (Tp) on the positive electrode side is set to 300 microseconds (μs), and the on-time (Tn) on the negative electrode side is set to 30 microseconds (μs) or more and less than 300 microseconds (μs). Can do. Regarding the off time, for example, the off time (Tpn) on the positive electrode side can be set to 100 microseconds (μs), and the on time (Tn) on the negative electrode side can be set to 100 microseconds (μs). The applied voltage can be, for example, ± 50 V. For example, the positive voltage (Vp) can be 20 V and the negative voltage (Vn) can be −20 V.

  In this case, since the duty ratio is different on the positive and negative sides, the timing of the pulse voltage applied to the target cell 100 is different on the positive and negative sides. Since the target cell 100 is not continuously charged with a charge with a biased polarity on either the positive or negative electrode, the introduced substance moves more efficiently and is more easily introduced while suppressing electrical damage. A situation will be formed.

  That is, in the case of a burst pulse in which the polarity of the reference pulse is continuously composed of a positive electrode or a negative electrode, energy due to the pulse voltage applied to the target cell 100 is amplified and electrical damage is likely to occur. Thus, in the asymmetric burst pulse according to this embodiment, the polarity of the reference pulse is alternately repeated with the positive electrode and the negative electrode, thereby suppressing electrical damage to the target cell 100 and introducing it more efficiently. A situation where a substance is easily introduced is formed.

  In addition, the asymmetric burst pulse according to the above-described embodiment can be configured by sequentially attenuating the applied charge amount of each reference pulse as shown in FIG. In this case, since the magnitude of the burst pulse given to the target cell 100 decays with time, the intensity of the burst pulse immediately before the subsequent high voltage pulse is applied is the initial value of the burst pulse application. Since it becomes smaller than the reference pulse, the difference between the high voltage pulse and the peak value of the burst pulse is maintained while maintaining the above-described effect that the burst pulse according to this embodiment gives to the target cell 100 and the introduced substance 110. The situation will be increased and the target cell will be more readily acceptable for the introduced substance.

  Next, after applying this burst pulse, a high voltage pulse having a higher voltage and a shorter time than this burst pulse is applied to the target cell 100 for one on time (S1: high voltage pulse application step). The high voltage pulse is preferably applied with the same polarity as that of the polarity side with a smaller amount of applied charge among the positive and negative reference pulses constituting the burst pulse. Thus, since the high voltage pulse is applied with a polarity different from the polarity of the charge charged in the target cell 100 by the burst pulse, the charge charged on the outer membrane of the target cell 100 by the burst pulse. As a result, the target cell is prevented from being excessively damaged by superimposing the charge of the opposite polarity to the high voltage pulse, and the target cell is further damaged by the application of the high voltage pulse. And more introduction substance can be introduced into the target cell.

  The on-time of the high voltage pulse according to the present embodiment is not particularly limited, but is preferably 15 nanoseconds (ns) to 200 nanoseconds (ns), and can be, for example, 100 nanoseconds (ns). . The voltage value of the high voltage pulse according to the present embodiment is not particularly limited, but is preferably 1 kV to 10 kV, and can be 3 kV, for example.

  For example, as shown in FIG. 3A, the asymmetric burst pulse and the high voltage pulse according to the present embodiment are separated from the high voltage pulse on the positive electrode side after a time interval (ΔT) after the application of the burst pulse. Application is made for one on-time on either pole side of the negative electrode side. The time interval (ΔT) is not particularly limited, but is preferably 10 microseconds or more, more preferably 1 to several hundred milliseconds, for example, 100 milliseconds (ms). it can. Below 1 millisecond, the tendency for electrical damage to increase increases.

  As described above, the burst pulse and the high voltage pulse according to the present embodiment damage the target cell 100 by applying the high voltage pulse to the target cell 100 after the burst pulse is applied. In addition, a large amount of the introduced substance 110 can be introduced into the target cell 100 at once. Although the mechanism that exerts this remarkable effect has not yet been elucidated in detail, an asymmetric burst pulse does not continuously charge a charge with a biased polarity to either the positive or negative electrode. The excessive electrical damage to the (outer membrane) is suppressed, while the introduced substance immersed in the solution is moved in either the positive or negative direction, so that the introduced substance is easily introduced. In this situation, when the high voltage pulse is applied for a short time, the action of moving the heavy introduction material 110 by the asymmetric burst pulse and the temporary action on the outer membrane by the high voltage pulse are performed. It is presumed that the introduction substance 110 is introduced into the target cell 100 at once without damaging the target cell 100 due to the formation of a proper hole. .

  In the present embodiment, the generation of two different pulses such as a burst pulse and a high voltage pulse can be realized by constructing a system having a circuit configuration as shown in FIG. 3B, for example. Can do. That is, the pulse control means 4 generates burst pulses based on the power supplied from the DC power supply 31 by switching control of RC discharge circuits (same polarity and reverse polarity) having two different polar outputs. The high voltage pulse generator 42 that generates a high voltage rectangular wave pulse with a Bloom line generator composed of a plurality of Bloom line lines by the power supplied from the unit 41, the DC high voltage power supply 32, and the control of the delay pulse generator The timing adjustment unit 43 that generates the burst pulse and the high voltage pulse with a delay of the time interval (ΔT), and the burst pulse and the high voltage pulse output from the burst pulse generation unit 41 and the high voltage pulse generation unit 42. And an output control unit 44 that outputs. When an electric pulse for generating a high voltage pulse is transmitted from the delay pulse generator to the bloom line generator, in order to suppress an electrical external influence that occurs when the bloom line generator operates, E / It is preferable to use optical fiber transmission using an O converter or the like.

(Second Embodiment)
In the substance introduction device according to the second embodiment, at least one of the counter electrode 22 and the counter electrode 23 in the first embodiment includes a pin-shaped or rail-shaped protrusion on the side facing the other electrode. Is. For example, as illustrated in FIG. 4, the protrusion is a pin-shaped pin-shaped protrusion 22 a or a rail-shaped rail-shaped protrusion 22 b. This protrusion increases the concentration (locality) of the discharge position due to the burst pulse and the high voltage pulse in the protrusion, and increases the concentration of the introduced substance immersed in the solution. By adjusting the amount of charge charged to the membrane, a large amount of introduced substance can be introduced into the target cell at once without damaging the target cell.

  In each of the above embodiments, the burst pulse (asymmetric burst pulse) is configured to maintain the zero level for a predetermined time while the polarity changes between positive and negative, but the potential value level maintained for the predetermined time is set to the zero level. Any potential value level can be used as long as it is an intermediate value of the changing positive and negative polarities.

(Third embodiment)
The substance introduction device according to the third embodiment applies a burst pulse in the subsequent stage after applying a high voltage pulse in the first embodiment or the second embodiment. That is, as shown in FIG. 5B, after the first burst pulse (previous burst pulse) is applied (S1), the high voltage pulse is applied (S2) in the high voltage pulse application step. Then, a subsequent burst pulse in which an asymmetric reference pulse that maintains a constant potential value level for a predetermined time while the polarity changes positively and negatively is repeated in the same cycle is applied to the target cell (S3: Subsequent burst pulse application step).

  In this way, a weak burst as a post-process of the high-voltage pulse is added to the target cell into which the introduction substance is introduced by applying the pre-stage burst pulse and the high-voltage pulse. Since the pulse is continuously applied, further electrical stimulation is continuously applied to the target cell to promote the introduction of the introduced substance, and the introduction of the introduced substance to the target cell is further ensured. Can be made.

  In this embodiment, as shown in FIG. 5 (b), two-stage burst pulses of a front-stage burst pulse and a rear-stage burst pulse are used. Burst pulses can be used. For example, the preceding burst pulse can be set to two or more stages and the latter burst pulse can be set to one stage. Conversely, the preceding burst pulse can be set to one stage and the preceding burst pulse can be set to two or more stages. You can also. Furthermore, both the front burst pulse and the rear burst pulse can be in two or more stages. In this way, since the configuration of the first burst pulse and the subsequent burst pulse can be freely set, the optimum burst pulse configuration can be selected according to the type of the introduced substance, and the introduction of the introduced substance can be made more efficient. it can.

(Fourth embodiment)
As shown in FIG. 5C, the substance introduction device according to the third embodiment is the first reference pulse that constitutes the burst pulse applied by the subsequent burst pulse application step in the third embodiment. Is a peak value of the last reference pulse constituting the preceding-stage burst pulse.

  As described above, since the target cell after the introduction substance is introduced, the electrical stimulation that is gentler than that of the preceding burst pulse is continuously given by the latter burst pulse. The introduction of the introduced substance is further stabilized, and the introduced substance can be more reliably introduced into the target cell.

  In each of the above embodiments, the burst pulse (asymmetric burst pulse) is configured to maintain the zero level for a predetermined time while the polarity changes between positive and negative, but the potential value level maintained for the predetermined time is set to the zero level. Any potential value level can be used as long as it is an intermediate value of the changing positive and negative polarities.

  EXAMPLES Examples will be described below to more specifically illustrate the features of the present invention, but the present invention is not limited to the following examples.

Example 1
Using the substance introduction apparatus shown in the first embodiment, the introduction substances are cycloheximide (CX) and actinomycin D (AD), and the spawning medaka eggs are placed in a solution in which these are mixed, respectively. In contrast, an asymmetric burst pulse and a high voltage pulse were applied, left in the same solution for about 2 hours, washed, and then observed. Change in survival rate and mortality due to the ratio of pulse width TP (ON time (Tp)) (= 300us) on the positive electrode side to pulse width TN (ON time (Tn)) (30us to 300us, no application) on the negative electrode side confirmed. Experimental conditions are shown below (parameters are the same as those described in FIG. 3 (a) above).

  Among the above experimental conditions, the waveforms of the burst pulse and the high voltage pulse in each case of TP: TN = 300 μs: 150 μs, TP: TN = 300 μs: 60 μs, and TP (300 μs) only: no TN are shown in FIG. (B) to (d). The obtained result is shown in FIG.

  In Fig. 6, abnormalities such as the proportion of eggs where death was confirmed by the third day after application of the burst pulse and high voltage pulse (death), and death / delay on the third day after the pulse application were confirmed. The percentage of eggs that were not found (normal), and the percentage of eggs that were confirmed to have abnormalities such as development delay on the third day after application of burst pulse and high-voltage pulse (abnormality) are shown (number of samples: 12 / condition) . In the figure, “control” indicates that a medaka egg in a solution not mixed with the introduction substance is simply applied with a burst pulse and a high voltage pulse (comparative example), “CX” indicates cycloheximide, “AD” Represents actinomycin D, and “TP: TN =” represents the on-time of each pole side described above with reference to FIG. “No VN” means that only VP is applied 100 times.

  From this result, in the control (control), when only the pulse on the positive side [VP (Vp) only (no VN (Vn))], the percentage of normal individuals was as low as 8%. It turned out that the influence was great. This is considered to be caused by an increase in the amount of charge (accumulation) on one side of the outer membrane of the individual. In addition, in the burst pulse, when the positive and negative pulses were set to the same ratio (TP: TN = 300μs: 300μs), there was almost no death of the individual. It was found that there was little impact. This is thought to be due to the fact that the amount of charge (accumulation) and discharge (discharge) generated in the outer membrane of the individual by the application of the burst pulse were the same. When the negative reference pulse (VN (Vn)) was applied, the average percentage of normal individuals was 78% (67% to 92%), and the effects of electrical damage were suppressed.

  Regarding the introduction of cycloheximide (CX), the percentage of normal individuals was 0%, and all individuals developed abnormalities (delays) or deaths mainly due to the introduction of CX. Furthermore, the shorter the on-time (pulse width; TN (Tn)) of the negative pulse (negative pulse), the mortality tended to increase (8% mortality at 300 μs, 75% at 30 μs). ). This is thought to be due to an increase in the amount of CX introduced. This is because the application of asymmetric burst pulses (TP ≠ TN) causes charge charges (accumulation) and discharges (discharges) to occur in the outer membrane of the individual with different amounts of charge, suppressing electrical damage to the individual. The introduction substance (CX) is moved in one direction in the solution by applying a relatively low voltage and long-term electrical energy by the burst pulse, and is introduced into the individual by the application of the subsequent high voltage pulse. This is thought to be caused by the increase of introduced substances (CX) (increased mortality due to CX).

  Regarding the introduction of actinomycin D (AD), 78% to 92% of individuals experienced abnormalities (delays in development) or death due to AD introduction. When the on-time (pulse width; TN (Tn)) of the negative pulse (negative pulse) was short (100 μs, 60 μs, 30 μs), the mortality rate exceeded 50%. From these results, the asymmetric burst pulse suppressed the electrical damage to the introduced substance and increased the amount of introduced substance.

(Example 2)
Using the substance introduction apparatus shown in the first embodiment, after application of an asymmetric burst pulse and a high-voltage pulse to a medaka egg after laying in the presence or absence of cycloheximide (CX) in the solution, The solution was allowed to stand for 2 hours, washed, and the subsequent state was observed. FIG. 6B shows the results when the positive-side voltage (Vp) (peak value), the negative-side voltage (Vn) (peak value) are 12 V, and the number of burst pulses (n) is 100, 200, and 400. ). From this result, it was found that good results were obtained when the number (n) of burst pulses in the asymmetric burst pulse was in the range of 100 to 200.

(Example 3)
Using the substance introduction apparatus shown in the first embodiment, the medaka eggs after egg laying are treated with bisphenol A (BPA) and 0 μm in concentrations of 5 μM and 50 μM in the presence or absence of asymmetric burst pulses and high voltage pulses. Washed after exposure to 37 nM estradiol for 2 hours. The following table shows the actual measurement values of bisphenol A in the in-vitro solution by gas chromatograph mass spectrometer (GC / MS) when bisphenol A is taken in with 50 μM of the external solution. By simply immersing a medaka egg in 50 μM bisphenol A for 2 hours, the amount of uptake was very small, and the concentration in the egg liquid was about 2 μM. However, by applying an asymmetric burst pulse and a high voltage pulse, about 90 μM The concentration of the inner solution increased dramatically, and bisphenol A could be taken into the medaka egg so that the concentration was equal to or higher than the concentration of the outer solution.

Example 4
Using the substance introduction apparatus shown in the first embodiment, the egg-laying medaka egg (the egg on the day of fertilization (day 0)) placed in a solution in which the introduction substance actinomycin D (AD) is mixed. ), An asymmetric burst pulse and a high voltage pulse were applied, left in the same solution for about 2 hours, washed, and the subsequent state was observed. As a comparative example, the presence or absence of a burst pulse, the presence or absence of actinomycin D (AD), and the case where the reference pulse is a burst pulse only on the positive electrode side were also carried out. confirmed. Experimental conditions are shown below (parameters are the same as those described in FIG. 3 (a) above).

The obtained result is shown in FIG. In FIG. 7A, a pulse applied to a fertilized egg in a solution not mixed with actinomycin D (AD) is pulsed to a fertilized egg in a solution mixed with “-AD” and actinomycin D (AD). The applied voltage is “+ AD”, that is, the following results are shown in order from the left side.
(No AD, only asymmetric burst pulse)
(No AD, pre-processing: asymmetric burst pulse, post-processing: high voltage pulse)
(No AD, only burst pulse consisting of the positive reference pulse only)
(With AD, asymmetric burst pulse only)
(With AD, preprocessing: asymmetric burst pulse, postprocessing: high voltage pulse)
(With AD, only burst pulse consisting of only positive reference pulse)

  From the obtained results, it was confirmed that actinomycin D was significantly introduced in the sample (with AD, pretreatment: asymmetric burst pulse, posttreatment: high voltage pulse). In addition, it was suggested that the electrical damage given to a fertilized egg is large in the example of only the burst pulse including only the reference pulse on the positive electrode side. On the other hand, it was confirmed that the introduction of actinomycin D was insufficient in the sample of the comparative example (with AD, only asymmetric burst pulse).

(Example 5)
Furthermore, by using the substance introduction apparatus shown in the first embodiment, bisphenol A (BPA) dissolved in dimethyl sulfoxide is diluted with a NaCl solution to prepare a bisphenol A (BPA) solution having a concentration of 5 μM, After laying eggs, medaka eggs were immersed, and bisphenol A (BPA) was introduced into medaka fertilized eggs by asymmetric burst pulses and high voltage pulses. Using the same procedure, the rate of occurrence of abnormalities was confirmed for each of the following chemical substances within 6 days after introduction into a medaka fertilized egg. As a reference example, the case of no introduction of chemical substances (control: only pulse application) was also performed.
Bisphenol A (BPA): 5μM
Bisphenol S (BPS): 5μM
Bisphenol C (BPC): 5μM
Bisphenol AF (BPAF): 5μM
Estradiol (E2): 0.37nM
Equilin: 0.037nM

The substance introduction apparatus was used under the following electrical conditions.
Positive side pulse width TP (on time (Tp)): 300μs
Negative side pulse width TN (ON time (Tn)): 30μs
Positive side voltage (Vp): 6V
Negative voltage (Vn): 6V
Off time (Tpn) on the positive side of the reference pulse of the burst pulse: 100μs
Off time (Tnp) on the negative side of the reference pulse of the burst pulse: 100μs
Number of burst pulse periods (n): 100
Time interval between burst pulse and high voltage pulse (ΔT1): 100 ms
High voltage pulse applied voltage: 3 kV
High voltage pulse application time: 100 ns

  The obtained result is shown in FIG. From the result of FIG. 7B, it was confirmed that the generation abnormality rate of the group (control) to which only the pulse was applied was 8.8%. On the other hand, the group that introduced chemical substances showed an increase in the incidence of abnormalities compared to the control group. Furthermore, in the group that introduced chemical substances, there was a clear difference in the abnormal incidence of medaka fertilized eggs for each chemical substance introduced. Thus, since the influence with respect to the initial generation of a medaka fertilized egg differed for every introduced chemical substance, it was confirmed that various chemical substances are certainly introduce | transduced inside the medaka fertilized egg. Moreover, since the influence of the medaka fertilized egg was different for each introduced chemical substance, it was confirmed that this substance introduction method can be used as an influence evaluation of various chemical substances.

(Example 6)
Next, GFP (Green Fluorescent Protein) was introduced into a medaka fertilized egg using the substance introduction apparatus shown in the first embodiment. The vector used was pEGFP-N3 (Clontech) at a concentration of 300 μg / ml. The fertilized egg on the day of sperm egg was immersed in a vector solution and pulsed under the following conditions (time: AM9: 20-AM10: 27).

Positive side pulse width TP (on time (Tp)): 300μs
Negative side pulse width TN (ON time (Tn): 30μs
Positive side voltage (Vp): 10.4 V
Negative voltage (Vn): 12.1 V
Off time (Tpn) on the positive side of the reference pulse of the burst pulse: 100μs
Off time (Tnp) on the negative side of the reference pulse of the burst pulse: 100μs
Number of burst pulse periods (n): 800
Time interval between burst pulse and high voltage pulse (ΔT1): 100 ms
High voltage pulse applied voltage: 3 kV
High voltage pulse application time: 100 ns

  FIG. 8 (a) shows a visible photograph and a fluorescence micrograph taken on fertilized eggs one day and two days after the introduction of the GFP vector. From the result of the fluorescence micrograph, it was confirmed that GFP was surely introduced into the fertilized egg since clear fluorescence appeared after 2 days, but no clear fluorescence was seen after 1 day. .

(Example 7)
Next, GFP was introduced into a medaka fertilized egg using the substance introduction device (including the first and second burst pulses) shown in the fourth embodiment (implementation time: AM 11:30) -AM12: 30). Differences in the implementation conditions from the above Example 6 are shown below.

Positive side voltage (Vp): 11 V
Negative side voltage (Vn): 26 V
Number of periods of preceding burst pulse (m): 400
Number of subsequent burst pulse cycles (n): 400
Time interval between burst pulse and high voltage pulse (ΔT1): 100 ms
Time interval between burst pulse and high voltage pulse (ΔT2): 100 ms

  FIG. 8 (b) shows a visible photograph and a fluorescence micrograph taken on fertilized eggs one day and two days after the introduction of the GFP vector. From the result of the fluorescence micrograph, clear fluorescence was observed after 1 day, and more clear fluorescence appeared after 2 days. From this, it was confirmed that GFP was introduced into the fertilized egg by additionally applying a subsequent burst pulse.

(Example 8)
Next, GFP was introduced into a medaka fertilized egg using the substance introduction apparatus (including the first and second burst pulses) shown in the fourth embodiment (implementation time: AM 9:20). -AM10: 27). Differences in implementation conditions from Example 6 are shown below.

Positive side voltage (Vp): 10.1 V
Negative voltage (Vn): 12.1 V

  FIG. 9 shows a visible photograph and a fluorescence micrograph taken on a fertilized egg one day after introduction of the GFP vector. From the result of the fluorescence micrograph, clear fluorescence already appeared after 1 day. From this, it was confirmed that GFP was surely introduced into the fertilized egg by applying the latter burst pulse that was gentler than the first burst pulse.

  FIG. 10 shows the ratio of fertilized eggs in which fluorescence due to GFP was confirmed on the first day after the introduction experiment as a result of the introduction experiment of the GFP vector under the following pulse conditions.

Introduced substance: GFP vector Target: medaka fertilized eggs Fertilized eggs, 1 group 48 sample pulse conditions The following 3 patterns / high voltage pulses only (3.2 kV, 100 ns)
・ High voltage pulse (3.2 kV, 100 ns) + 400 asymmetric burst pulse latter stage [(+8 V, 300 μs) + (− 8 V, 30 μs)]
200 asymmetric burst pulses before 200 [(+ 8V, 300 μs) + (− 8 V, 30 μs)] + high voltage pulse (3.2 kV, 100 ns) +200 after asymmetric burst pulses [(+8 V, 300 μs) + (− 8 V, 30μs)]

  From the experimental results, it was found that the introduction of a substance in which a burst pulse was added before and after the high voltage pulse (the fourth embodiment) was effective for introducing GFP.

Example 9
Next, actinomycin D (AD) was introduced into a medaka fertilized egg using the substance introduction apparatus (including the first and second burst pulses) shown in the fourth embodiment. The implementation conditions are asymmetric burst pulse pre-stage 400 [[+5 V, 300 μs) + (− 5 V, 30 μs)] + high voltage pulse (3.6 kV, 100 ns).

  FIG. 11 shows the status of each fertilized egg on the third day after the introduction experiment of actinomycin D (AD) by applying pulses to the egg on the day of fertilization (day 0) and the egg on the day after fertilization (day 1). "-AD" means a pulse applied to a fertilized egg in a solution not mixed with actinomycin D (AD), "+ AD means a pulse applied to a fertilized egg in a solution mixed with actinomycin D (AD)" “It was From the results, by using this substance introduction device, actinomycin D (AD) while suppressing the electrical influence on the fertilized egg for the egg on the day of fertilization (day 0) and the egg on the day after fertilization (day 1) It was confirmed that was introduced.

(Example 10)
A warfarin sodium introduction experiment was conducted using the substance introduction device under the following conditions.

Introduced substance: Anticoagulant component Warfarin sodium (0, 1, 10, 100 ppm)
Target: Medaka fertilized egg Fertilization day egg, 1 group 12 sample pulse conditions 1 pattern / asymmetric burst pulse 100 preceding stage [(+7 V, 300 μs) + (−7 V, 30 μs)] + high voltage pulse (3 kV, 100 ns) ]

  FIG. 12 shows the results of a warfarin sodium introduction experiment conducted using this substance introduction apparatus. As a result of observing 6 days after pulse application (before hatching) and after hatching, there are those that show abnormalities such as blood circulation and stagnation in individuals before hatching, and abnormalities such as abdominal (heart) hypertrophy and spinal curvature in individuals after hatching confirmed. Looking at the percentage of these abnormalities, the incidence of abnormalities in fertilized eggs pulsed in a solution of 100 ppm of warfarin sodium was prominently high at approximately 82%, and warfarin sodium was significantly introduced. It was confirmed.

(Example 11)
Under the same conditions as in Example 10, the following introduction experiment of loxoprofen sodium dihydrate was conducted in place of warfarin sodium.

  Introduced substance: Non-steroidal anti-inflammatory drug component Loxoprofen sodium dihydrate (0, 0.1, 1, 10, 100 ppm)

  A visible photograph taken on a fertilized egg after the introduction of loxoprofen sodium dihydrate is shown in FIG. As described above, a plurality of individuals exhibiting abnormal angiogenesis (blood circulation) before hatching and abnormal development such as abdominal (heart) hypertrophy and spinal curvature after hatching were confirmed.

DESCRIPTION OF SYMBOLS 100 Target cell 110 Introduction | transduction substance 120 Solution 21 Storage means 22 Counter electrode 22a Pin type protrusion 22b Rail type protrusion 23 Counter electrode 3 DC power supply 31 Burst pulse power supply 32 High voltage pulse power supply 4 Pulse control means 41 Burst pulse generation part 42 High Voltage Pulse Generation Unit 43 Timing Adjustment Unit 44 Output Control Unit

Claims (12)

In a substance introduction method for introducing an introduction substance into a target cell,
A burst pulse in which an asymmetric reference pulse, which is maintained at a constant potential level for a predetermined time while the polarity changes positively and negatively, is repeated in the same cycle, is applied to a target cell immersed in a solution containing an introduced substance. A burst pulse applying step to be applied;
A high-voltage pulse applying step of applying a high-voltage pulse having a higher voltage and a shorter time than the reference pulse constituting the burst pulse to the target cell after application by the burst pulse application step; Substance introduction method characterized by including. The substance introduction method according to claim 1,
The substance introduction method, wherein the reference pulse constituting the burst pulse has different peak values between positive and negative polarities. In the substance introduction method according to claim 1 or 2,
The substance introduction method, wherein the reference pulse constituting the burst pulse has a different duty ratio between positive and negative polarities. In the substance introduction method according to any one of claims 1 to 3,
The substance introduction method, wherein the burst pulse is configured by sequentially attenuating the applied charge amount of each reference pulse. In the substance introduction method according to any one of claims 1 to 4,
The substance introduction method, wherein the high-voltage pulse is applied with the same polarity as that of a polarity side with a small amount of applied charge among positive and negative reference pulses constituting the burst pulse. In the substance introduction method according to any one of claims 1 to 5,
The reference pulse constituting the burst pulse has an on-time of the order of microseconds and an applied voltage of the order of several tens to several hundreds V.
The high voltage pulse has an on-time of nanosecond order and an applied voltage of kV order. In the substance introduction method according to any one of claims 1 to 6,
The substance introduction method, wherein the applied charge amount of each positive and negative reference pulse constituting the burst pulse has a ratio of 10: 1 to 1:10 between each positive and negative. In the substance introduction method according to any one of claims 1 to 7,
After the high voltage pulse is applied by the high voltage pulse application step, a burst pulse in which an asymmetric reference pulse that maintains a constant potential value level for a predetermined time while the polarity changes between positive and negative for a predetermined period, And a subsequent burst pulse applying step for applying to the target cell. In the substance introduction method according to claim 8,
The peak value of the first reference pulse constituting the burst pulse applied by the subsequent burst pulse applying step is less than the peak value of the last reference pulse constituting the burst pulse applied by the burst pulse applying step corresponding to the preceding stage. A substance introduction method characterized by In the substance introduction device for introducing the introduction substance into the target cell,
A container containing the solution containing the introduced substance and the target cell;
A pair of counter electrodes disposed around the housing means;
A DC power supply for supplying a DC current for generating a voltage to be applied between the counter electrodes;
A burst pulse in which an asymmetric reference pulse, which is maintained at a constant potential value level for a predetermined time while the polarity changes positively and negatively based on a direct current supplied from the direct current power source, is repeated in the same period, and the burst pulse And a pulse control means for controlling the application of a high-voltage pulse having a higher voltage and a shorter time than the reference pulse constituting the target cell to the target cell. The substance introduction device according to claim 10,
The pulse control means comprises:
A burst pulse generator for generating the burst pulse;
A high voltage pulse generating unit for generating the high voltage pulse;
A substance introduction apparatus comprising: a timing adjustment unit that adjusts the timing of applying the burst pulse and the high voltage pulse. The substance introduction device according to claim 10 or 11,
At least one of the counter electrodes is provided with a pin-shaped or rail-shaped projection on the side facing the other electrode.