JP4187060B2 - Cell processing method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
In the present invention, a treatment for transforming animal and plant cells for the purpose of improving the variety of animals and plants, promoting growth, etc., is performed on the living bodies of animals and plants excluding humans , or on suspensions of cells taken out of living bodies of animals and plants including humans ( The present invention relates to a treatment method performed by energization operation on a cell-containing system).
[0002]
[Prior art]
In recent years, in addition to traditional mating, cell treatment (cell manipulation) that adds treatment to cells themselves, such as cell fusion between different species or introduction of other species into cells, has become popular as a means for improving the variety of animals and plants. It is becoming. The cell treatment is usually performed in such a manner that an energization operation is applied to the cell-containing system.
[0003]
In cell fusion, high-frequency energization is first applied to a cell-containing system, and the cells are dielectrophoresed in the system to produce a state where the cells are closely arranged in a daisy chain (pearl chain), and then to the above system. High voltage pulse energization is performed through a series of energization operations in which a cell membrane in a region where cells are in contact with each other is temporarily destroyed and opened in a window shape. Thus, after the above operation, the cell membrane is repaired in such a manner that the window frames of adjacent cell openings are joined together, and two or more cells are integrated (fused). Therefore, if there are multiple types of cells in the system, cell fusion between different species will occur.
[0004]
In the case of gene transfer, sudden application of high-voltage pulse to the cell-containing system causes partial destruction of the cell membrane, creating an opening or weakened part (electroporation), between the inside and outside of the cell Facilitates the distribution or transmission of In addition, the entry / exit of charged substances such as other types of DNA through the opening or the like is promoted by electrophoresis or electric permeation by direct current application to the cell-containing system. The opening and the like are also self-repaired thereafter. The above treatment via electroporation is also useful for extraction of intracellular product.
[0005]
The energization operation for cell processing such as cell fusion and gene transfer is performed by an electric field induction method in which an electrode pair is placed inside or outside the cell-containing system and an electric field is generated by applying a voltage to the electrode pair. Has been done. Therefore, when the energization operation is applied to the living body of an animal or plant itself, the arrangement of the electrode pair is as follows: (1) the electrode is brought into close contact with the surface of the living body, or (2) the needle-like or blade-like electrode is placed from the surface of the living body. This can be done either by inserting into a desired site in the living body or by (3) disposing the electrode by once exposing the desired site in the living body by incision surgery. Therefore, in the form (1), the electric flux is formed so as to spread from the electrode in a spindle shape, so that it is difficult to form an electric field concentrated on the desired part. In other words, the electric field of the desired condition is applied to the desired part. If it tries to form, it will exceed the said desired conditions with respect to another site | part, especially an epidermis (skin etc.), and will bring the electricity load which is originally unnecessary. Further, in the form (2) or (3), although it is easy to form a concentrated electric field at a desired site, the living body is subjected to a greater invasion than the form (1). In other words, it is difficult to apply cell treatment without causing a deviation from the normal state of the biological function, and then continue the growth experiment itself, and there are extremely limited subjects to which application can be attempted. there were.
[0006]
[Problems to be solved by the invention]
The present invention has been made in view of the above circumstances, and the electric field formation for the high voltage pulse energization or the like can be performed without using an electrode, and the above-mentioned limitation derived from the use of the electrode can be overcome. It is an object to provide a cell treatment method.
[0007]
[Means for Solving the Problems]
The cell treatment method of the present invention, which has been made to solve the above-mentioned problems, is an electric field applied to a system containing either one of a suspension of a cell group taken out of a living animal or plant, or a living organism of a living animal excluding humans. Is a cell processing method for causing a cell to move in the system and causing a change in the cell membrane, or to promote a change in the intracellular composition, and facing the system containing the cell to the electromagnetic coil Is applied to the electromagnetic coil so that the current waveform in the current rise period and the current waveform in the current fall period are asymmetric in time series, and the plus side and the minus side have asymmetric polarities. A pulse voltage is induced in a system containing the cells.
[0008]
That is, according to the present invention, an energization operation for cell treatment is performed by forming an electric field in the system by an electromagnetic induction method using an electromagnetic coil instead of an electrode, and an energization polarity required for pulse energization. This can be done in the form of having the above, and the limit derived from the use of the electrode is overcome and the problem of the present invention is solved. Incidentally, the voltage induced to the load by the electromagnetic induction method is usually an alternating current, and has a substantially uniform amplitude on both the positive and negative sides, that is, has no polarity.
[0009]
In the electromagnetic induction method, since an electromagnetic coil is arranged near an object to be energized and the object can be energized by energizing the coil, no electrode arrangement is required. That is, (b) cell treatment can be performed without invasion of the living body as described above, and (b) a strong action on other parts rather than the desired part, and further, (C) By forming the electromagnetic coil shape and arrangement, it is possible to form a magnetic flux aiming at a desired part and thus even an electric flux.
[0010]
The energization of the electromagnetic coil in the present invention, that is, the pulse energization (hereinafter referred to as “asymmetric energization”) in which the current waveform in the current rising period and the current waveform in the current falling period are asymmetric in time series is, for example, A) Energization in which the current value suddenly rises and slowly decreases, or (b) Energization in which the current value rises slowly and rapidly decreases. An energization mode in which there is a holding period in which the current value hardly changes between the rising period in which the current value rises and the falling period in which the current value decreases is also applicable to the present invention.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The example of arrangement | positioning of the electromagnetic coil to the cell containing system in this invention is shown in FIG. In the example of FIG. 1, the living body 1 of a mouse into which another kind of DNA has been introduced by injection is filled as a cell-containing system, and an 8-shaped multi-turn coil 2 is used as the electromagnetic coil, which is the body surface of the DNA introduction site. It is arranged. The smaller the distance between the living body 1 and the coil 2, the more efficiently the magnetic field can be formed in the system. For example, an electrical insulation sheet (cloth material or plastic sheet having a thickness of about 0.1 to 2 mm) is interposed instead of the spacer. Thus, the state in which the coil is in close contact with the living body 1 may be held with the aid of mechanical support means. As a cell-containing system, a non-conductive container, a cell group suspension accommodated in a pipe line, or a cell group suspension being transferred can naturally be targeted. In addition, the coil 2 is shaped like a figure 8 and is arranged in a face-to-face manner by concentrating the magnetic flux in the living body of the part facing the part 2a where the windings overlap, and concentrating on this part. In order to generate an electric field, the shape and arrangement of the electromagnetic coil used in the present invention are not limited to the above-described form, and a solenoid coil (spiral cylinder) is arranged so as to surround the living body, and the magnetic flux is applied to the entire living body. Other forms may be appropriately employed depending on the purpose.
[0012]
Next, a method of applying a pulse to the electromagnetic coil in the present invention will be described. FIG. 2 shows an example of an output circuit for performing the asymmetric energization, wherein 3 is a DC power supply, 4 is an ON / OFF switch, 5 is a flywheel diode, 6 is a resistance for adjusting an attenuation factor (resistance value R ₁ ), 7 and 7 'are output terminals. Inductance of the induction coil 2 is L, the resistance value is assumed to be R _2. The switch 4 can be composed of, for example, a switching transistor and a timer that can output a YES / NO signal in a desired time-series mode. Any mechanism can be used as long as it can perform ON / OFF operation.
[0013]
In the circuit of FIG. 2, when the switch 4 is turned ON / OFF as shown in FIG. 3 (a), the coil 2 has a current rising waveform and a falling period as shown in FIG. 3 (b). A pulse current with an asymmetric waveform in time series flows, and a pulse voltage with polarity as shown in Fig. 3 (c) (asymmetric on the plus and minus sides) is induced in the cell-containing system facing the coil. Is done.
[0014]
Here, as the voltage E of the DC power supply increases, the steepness of the rising edge (θ ₁ tangent in FIG. 3, that is, tan θ ₁ ) in FIG. 3B becomes larger, and as a result, the positive side of FIG. The magnitude of the peak voltage | E _p1 | On the other hand, the sum R = R ₁ + R _{2 of} the attenuation rate adjusting resistor 6 and the internal resistance of the coil 2 (the minimum value R ₂ is obtained when the resistor 5 is omitted and the diode 5 is directly connected), and the inductance L of the coil 2 The larger the ratio R / L, the greater the steepness of attenuation in the current falling period of FIG. 3B (for example, the tangent of θ _{2 in} the figure, that is, tan θ ₂ = can be expressed by the average gradient until the current half-life), As a result, the magnitude | E _p2 | of the negative side peak voltage in FIG. The polarity of the induced voltage in FIG. 3C is expressed by the ratio or difference between the above | E _p1 | and | E _p2 |, so that the DC input voltage E is increased and the resistance value R ₁ of the attenuation factor adjusting resistor is increased. The smaller the is, the more prominent the polarity of the induced voltage is.
[0015]
The degree of polarity of the induced voltage is desirably set to about 3 to 10 as a ratio of | E _p1 | / | Ep ₂ |. This is because when the ratio is 3 or more, the DC bias level (| E _p1 | − | E _p2 |) / 2 in the induced pulse voltage is ₂ or more and the negative peak level | E _p2 | The larger the ratio, the better the purpose of the present invention. On the other hand, in order to increase the ratio | E _p1 | / | E _p2 |, the steepness tanθ ₂ of the waveform during the coil current falling period (see FIG. 3). (The following is also required) to be small (slowly attenuated), and accordingly, it is necessary to take a longer time for the switch 4 to perform the OFF operation, and the time ratio of the ON operation, that is, the appearance cycle of the induced pulse voltage is generated. This is because it is difficult to set | E _p1 | / | E _p2 | ratio to a level exceeding 10.
[0016]
The above | E _p1 | / | E _p2 | ratio can be optimized by adjusting the DC input voltage E and the resistance value R ₁ of the attenuation factor adjusting resistor as described above. In addition, regarding the ON / OFF operation mode of the switch 4 shown in FIG. 3A, the ON operation time t ₁ and the OFF operation time in the drawing are optimized in order to optimize the ratio | E _p1 | / | E _p2 | Basically, since t ₂ and the ratio t ₁ / t ₂ are limited, there is a need to experimentally optimize the appearance cycle of the induced pulse voltage in each cell treatment trial. Specifically, it should be selected for each cell treatment trial.
[0017]
In the above description, | E _p1 | (plus side) is larger than | E _p2 | (minus side). However, this is an expedient setting for explanation and is actually an energization target. What is necessary is just to set the connection polarity of the circuit including a coil so that the main direction of electricity supply in a cell containing system which is can be made into a desired direction. Alternatively, as a suitable range of the | E _p1 | / | E _p2 | ratio, in addition to 3 to 10, 0.1 to 0.3 (reciprocal of 3 to 10) corresponding to the case where E _p2 is larger may be added. Also good.
[0018]
As described above, by energizing the electromagnetic coil by the output circuit illustrated in FIG. 2, high voltage pulse energization having polarity can be performed in the target cell-containing system, for the cell fusion or gene introduction. Electroporation, electrophoresis, etc. can be performed. In order to confirm the aspect of the above-mentioned pulse energization, an energization experiment was performed using the equivalent model circuit shown in FIG. 4, and the result shown in FIG. 5 was obtained. In FIG. 4, 8 is the equivalent resistance (1 kΩ) as seen in the cell-containing system, and 9 is a secondary coil for equivalently reproducing the electromagnetic induction effect on the cell-containing system. 5A shows the waveform of the pulse current flowing through the coil 2, and FIG. 5B shows the waveform of the pulse voltage measured at both ends of the equivalent resistor 8. FIG. As shown in FIG. 5, a pulse voltage of | E _p1 | / | E _p2 | = 5: 1 is induced by asymmetric pulse energization of the coil 2. Incidentally, when a normal pulse energization symmetric in time series is applied to the coil 2 as shown in FIG. 6 (a), the normal AC pulse having no polarity shown in FIG. 6 (b) is induced. Yes.
[0019]
As described above, high-voltage pulse energization for cell fusion, electroporation, etc. is performed by generating a pulse voltage with polarity in the target system. Decomposition takes place, and changes in the composition of the system and by-products such as gas generated in accordance with the change cause changes in the system itself and hinder the performance of the treatment. It is also known that this problem can be avoided by energizing while appropriately switching the polarity of energization. Accordingly, in the present invention, for example, the circuit shown in FIG. 7 is operated as shown in FIG. 8 (a) (1) to (4), and the coil is energized in the current mode illustrated in FIG. 8 (b). Thus, it is possible to induce a pulse voltage in which the polarities are sequentially switched as shown in FIG.
[0020]
High-frequency energization (high-frequency alternating current energization) for forming the pearl chain, which is one of energization operations for cell fusion, can also be performed by polarity switching energization within the scope of the present invention. However, since the high-frequency energization can be performed by inputting the output of a normal high-frequency oscillator to the electromagnetic coil 2, any means may be selected according to the situation.
[0021]
Note that various operations other than the energization operation in the cell treatment method of the present invention may be performed by techniques according to conventional cell treatment methods.
[0022]
【The invention's effect】
As described above, the present invention provides cells by high-pressure pulse energization or the like to a system containing either a suspension of a cell group taken out of a living animal or plant, or a living organism of a plant or animal excluding humans. In the cell processing method that performs fusion and gene transfer, the electric field formation for energization is performed not by the electric field induction method but by the electromagnetic induction method, and the electric field having polarity is formed even though the electromagnetic induction method is used. Is. That is, cell treatment without using an electrode has become possible, and the limitations (eg difficult to apply to the living body itself) derived from the presence of the electrode in conventional cell treatment methods have been overcome.
[0023]
Cell treatment has changed as a means of improving breeds of animals and plants, as compared to traditional means of crossing. The scope of application of cell treatment has been greatly expanded by the present invention, and the application of animals and plants other than humans to living organisms themselves is greatly contributing to future animal husbandry, agriculture, and drug manufacturing businesses. Is.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of arrangement of electromagnetic coils in a cell-containing system.
FIG. 2 is a block diagram showing an example of an output circuit for performing asymmetric energization.
3 is a waveform showing the operation and output in the output circuit of FIG. 2, where (a) shows the operation timing waveform of the switch 4, (b) shows the current waveform flowing through the coil, and (c) shows in the cell-containing system. The wave form diagram which shows the induced pulse voltage waveform, respectively.
4 is a block diagram showing an example of an equivalent model circuit of the output circuit of FIG. 2. FIG.
5 is a waveform diagram of a pulse voltage measured by a pulse current flowing through the coil 2 of the circuit shown in FIG. 4 and an equivalent resistance 8. FIG.
FIG. 6 is a waveform diagram obtained by normal pulse energization.
FIG. 7 is a block diagram of another example of an output circuit that performs asymmetric energization.
8 is a waveform showing the operation and output of the output circuit of FIG. 7, where (a) shows the waveform of the operation timing, (b) shows the pulse energization waveform, and (c) shows the waveform of the induced pulse voltage. FIG.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Living body 2 Electromagnetic coil 3 Circuit 4, 4 'Input terminal 6 Secondary coil 5 Switch 7, 7' Output terminal 8 Resistance element

Claims (3)

An electric field is applied to a system containing either a suspension of cells taken out of a living animal or plant, or a living body of an animal or plant excluding humans, and the cell is moved within the system to form a cell membrane. A cell treatment method for causing a change or promoting a change in intracellular composition, wherein an electromagnetic coil is disposed opposite to a system containing the cell, and the electromagnetic coil has a current rising period. Conducting pulse energization so that the current waveform and the current waveform in the current fall period are asymmetric in time series, and inducing a pulse voltage having an asymmetric polarity on the plus side and the minus side in the system containing the cells A cell processing method. The cell processing method according to claim 1, wherein a ratio between a positive side peak voltage and a negative side peak voltage in a pulse voltage induced in a system including the cells is 3 to 10.   The cell according to claim 1 or 2, wherein the polarity of the pulse voltage induced in the system including the cell is switched in accordance with the switching by performing pulse energization to the induction coil while switching the polarity. Processing method.

Images