CN112980673A - High-frequency pulse magnetic field induced cell magnetic perforation device and method - Google Patents

High-frequency pulse magnetic field induced cell magnetic perforation device and method Download PDF

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CN112980673A
CN112980673A CN202110133475.8A CN202110133475A CN112980673A CN 112980673 A CN112980673 A CN 112980673A CN 202110133475 A CN202110133475 A CN 202110133475A CN 112980673 A CN112980673 A CN 112980673A
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米彦
代璐健
许宁
陈嘉诚
郑伟
马驰
李政民
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Chongqing University
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Abstract

The invention discloses a high-frequency pulse magnetic field induced cell magnetic perforation device and a method, wherein the device comprises a high-voltage direct-current power supply, a pulse capacitor, a switch driving device, a solid-state switch group and a magnetic field coil; the method comprises the following steps: 1) building a high-frequency pulse magnetic field induced cell magnetic perforation device; 2) placing an object to be processed in a target action area of the magnetic field coil; 3) presetting pulse parameters; 4) the high-voltage direct-current power supply charges the energy storage capacitor; 5) the FPGA module generates a switch control electric signal based on a preset pulse parameter; 6) the optical fiber receiver transmits the switch control electric signal to the driving chip; 7) the driving chip controls the on-off of the solid-state switch group; the pulse capacitor sends excitation pulse to the magnetic field coil; 8) the magnetic field coil generates a magnetic field to induce the object to be treated in the target action region to generate magnetic cell perforation. Compared with the traditional cell membrane electroporation technology, the magnetic electroporation technology can induce cell membrane electroporation in a non-contact, high-efficiency and non-invasive manner.

Description

High-frequency pulse magnetic field induced cell magnetic perforation device and method
Technical Field
The invention relates to the field of cell perforation, in particular to a high-frequency pulse magnetic field induced cell magnetic perforation device and a method.
Background
The pulsed electric field induced cell electroporation is a new type of biotechnology and has been widely used in the field of bioengineering. However, the conventional electroporation method has disadvantages in that it must be in direct contact with cells or in contact with a cell solution during the electroporation process, and has problems of low efficiency, poor electrical safety, etc.
Disclosure of Invention
The invention aims to provide a high-frequency pulse magnetic field induced cell magnetic perforation device, which comprises a high-voltage direct-current power supply, a pulse capacitor C, a switch driving device, a solid-state switch group, a magnetic field coil and a discharge resistor R for protecting a circuit to stably work;
the high-voltage direct-current power supply charges a pulse capacitor C;
the pulse capacitor C sends excitation pulse to the magnetic field coil through the solid-state switch group;
the switch driving device controls the on-off of the solid-state switch group to control the duration and the number of the excitation pulses;
the switch driving device comprises an FPGA module, an electro-optical converter, a switch power supply and a turn-off driving module with negative pressure;
the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module;
the FPGA module sends a switch control electric signal to the electro-optical converter;
the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
the switching-off stable drive with the negative pressure comprises a DC-DC isolation module, an optical fiber receiver and a drive chip;
the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group is controlled.
The solid state switch bank comprises N solid state switches; n is more than or equal to 2.
The N solid-state switches are arranged at intervals in the circumferential direction, wherein one ends of all the solid-state switches are connected to one point and recorded as a circle center O.
The magnetic field coil generates a magnetic field after receiving the excitation pulse, and the region of the magnetic field distribution is marked as a target action region;
an object to be treated is placed in the target action area;
and the object to be treated generates cell magnetic perforation under the action of a magnetic field.
And the FPGA module controls the on-off of the MOSFET switch group so as to control the duration and the number of the excitation pulses.
The equivalent circuit structure of the high-frequency pulse magnetic field induced cell magnetic perforation device is as follows:
recording that one end of a high-voltage direct-current power supply where a positive electrode is located is A, and one end of a negative electrode is B;
the A end is connected with the B end after being connected with the pulse capacitor C in series; recording the end of the pulse capacitor C which is not directly connected with the end A as D;
the A end is sequentially connected with the solid switch group, the magnetic field coil and the discharge resistor R in series and then grounded;
the A end is sequentially connected with the solid-state switch group, the magnetic field coil and the discharge resistor R in series and then connected with the B end;
the A end is sequentially connected with the solid-state switch group, the magnetic field coil and the discharge resistor R in series and then connected with the D end.
The method for inducing the cell magnetic perforation device based on the high-frequency pulse magnetic field comprises the following steps:
1) building a high-frequency pulse magnetic field induced cell magnetic perforation device;
2) placing an object to be processed in a target action area of the magnetic field coil;
3) presetting pulse parameters;
4) the high-voltage direct-current power supply charges the energy storage capacitor C; the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module; the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
5) after the energy storage capacitor C is charged, the FPGA module generates a switch control electric signal based on a preset pulse parameter and transmits the switch control electric signal to the electro-optical converter;
6) the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
7) the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
8) the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group is controlled;
the pulse capacitor C sends excitation pulse to the magnetic field coil through the solid-state switch group;
9) the magnetic field coil generates a magnetic field after receiving the excitation pulse, so that the object to be processed in the target action region is induced to generate magnetic cell perforation.
It is worth to say that the invention helps to induce the magnetic perforation of the cells at lower pulse magnetic field amplitude by the cumulative effect of the pulse by increasing the frequency of the pulse magnetic field to a high frequency.
According to the invention, specific experiment time is selected for target cells, and then the cells in the pulsed magnetic field are all adherent cells during experiment through selecting the experiment time. After the target cells are completely attached to the wall in the pore plate, a pulsed magnetic field with corresponding parameters is applied to induce the cells to generate magnetic perforation.
The technical effect of the invention is undoubted, and the invention can reach the critical condition required by the magnetic perforation of the cell under non-contact condition, thereby inducing the cell membrane to generate the perforation effect more efficiently and non-invasively.
Drawings
FIG. 1 is a schematic diagram of the magnetic perforation effect induced by high-frequency pulsed magnetic field;
FIG. 2 is a schematic block diagram of a high frequency pulse generator;
FIG. 3 is a schematic block diagram of high frequency pulse generator solid state switching pulse formation;
FIG. 4 is a block diagram of a solid state switch bank of the high frequency pulse generator;
FIG. 5 is a diagram of a single pulse waveform of the generator;
FIG. 6 is a graph showing the effect of different magnetic field amplitudes on the proportion of PI-positive cells;
FIG. 7 is a graph showing the effect of different pulse counts on the proportion of PI-positive cells;
FIG. 8 is a graph of the effect of different magnetic field amplitudes on the proportion of cells undergoing perforation;
FIG. 9 is a graph of the effect of different magnetic field amplitudes on the proportion of cells undergoing perforation.
FIG. 10 is an equivalent circuit diagram of a high-frequency pulsed magnetic field induced cell magnetic perforation apparatus;
in the figure, a solid-state switch group 1 and a magnetic field coil 2 are shown.
Detailed Description
The present invention is further illustrated by the following examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention and the scope of the invention is covered by the present invention according to the common technical knowledge and the conventional means in the field.
Example 1:
referring to fig. 1 to 5 and 10, the high-frequency pulse magnetic field induced cell magnetic perforation device comprises a high-voltage direct-current power supply, a pulse capacitor C, a switch driving device, a solid-state switch group 1, a magnetic field coil 2 and a discharge resistor R for protecting a circuit to stably work;
the high-voltage direct-current power supply charges a pulse capacitor C;
the pulse capacitor C sends excitation pulse to the magnetic field coil 2 through the solid-state switch group 1;
the switch driving device controls the on-off of the solid-state switch group 1 to control the duration and the number of excitation pulses;
the switch driving device comprises an FPGA module, an electro-optical converter, a switch power supply and a turn-off driving module with negative pressure;
the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module;
the FPGA module sends a switch control electric signal to the electro-optical converter;
the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
the switching-off stable drive with the negative pressure comprises a DC-DC isolation module, an optical fiber receiver and a drive chip;
the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group 1 is controlled.
The solid-state switch group 1 comprises N solid-state switches; n is more than or equal to 2.
The N solid-state switches are arranged at intervals in the circumferential direction, wherein one ends of all the solid-state switches are connected to one point and recorded as a circle center O. The center O is connected with the capacitor C, and the other end of the solid-state switch is connected with the magnetic field coil.
The magnetic field coil 2 generates a magnetic field after receiving the excitation pulse, and the region of the magnetic field distribution is marked as a target action region;
an object to be treated is placed in the target action area;
and the object to be treated generates cell magnetic perforation under the action of a magnetic field.
And the FPGA module controls the on-off of the MOSFET switch group so as to control the duration and the number of the excitation pulses.
The equivalent circuit structure of the high-frequency pulse magnetic field induced cell magnetic perforation device is as follows:
recording that one end of a high-voltage direct-current power supply where a positive electrode is located is A, and one end of a negative electrode is B;
the A end is connected with the B end after being connected with the pulse capacitor C in series; recording the end of the pulse capacitor C which is not directly connected with the end A as D;
the A end is sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series and then grounded;
the A end is sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series and then connected with the B end;
the A end is connected with the D end after being sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series.
Example 2:
referring to fig. 1 to 5 and 10, the high-frequency pulsed magnetic field induced cell magnetic perforation device includes a high-voltage dc power supply, a pulse capacitor, a solid-state switch set, a discharge resistor, and a magnetic field coil. The positive pole of high voltage direct current power supply is connected with the positive pole of the pulse capacitor through a lead, one end of the solid-state switch group is connected with the positive pole of the pulse capacitor through a copper sheet, the other end of the solid-state switch group is connected with one end of the magnetic field coil through a copper sheet, the other end of the magnetic field coil is connected with one end of the discharge resistor through a copper sheet, and the other end of the discharge resistor is directly connected with the negative pole of the pulse capacitor and is connected with the negative pole of the high voltage direct current power supply.
The high-speed solid-state switch group comprises N solid-state switches with the same type, and the number N can be designed by the required pulse amplitude and the rated current of a single solid-state switch; each solid-state switch is connected in parallel in a fan-shaped symmetrical arrangement mode, the electric paths of the solid-state switches are the same while a high-frequency pulse magnetic field is generated, and the current equalizing effect is achieved.
The stable drive with the negative pressure turn-off function comprises a switching power supply, a DC-DC isolation module, an optical fiber receiver and a drive chip; the switching power supply is used as a power supply and connected with the DC-DC isolation module, the DC-DC isolation module generates positive power supply voltage to supply power to the optical fiber receiver, the DC-DC isolation module generates positive and negative power supply voltage to supply power to the driving chip, and the driving chip generates positive and negative bipolar driving signals to control the on-off of the solid-state switch.
The high-frequency pulse magnetic field generating device generates a magnetic field waveform in a target area of the magnetic field coil, wherein the waveform is a high-frequency (hundred kilohertz) pulse, the amplitude of the high-frequency pulse magnetic field is adjusted by the high-voltage direct current power supply, and the pulse width, the repetition frequency and the pulse number of the high-frequency pulse magnetic field are adjusted by the bipolar driving signal.
Example 3:
referring to fig. 2, a high frequency pulse generator includes a high voltage dc power supply, a pulse capacitor, a solid state switch bank, a discharge resistor, and a field coil. The high-voltage direct-current power supply converts 220V alternating current into high-voltage direct-current voltage, and after the high-voltage direct-current power supply charges the pulse capacitor to the required voltage, the high-voltage direct-current power supply controls the on-off of the solid-state switch group to generate pulse heavy current in the discharge loop, so that a pulse magnetic field is generated under the action of the magnetic field coil. In fig. 2, the discharge resistor is directed to the field coil, and it is shown that the discharge resistor stabilizes the voltage of the pulse in the circuit, and the stabilized pulse is input to the field coil.
Referring to fig. 3, the solid-state switch pulse forming part comprises a switching power supply, a DC-DC isolation module, an optical fiber receiver and a driving chip. The switch power supply is used as the input end of the DC-DC isolation module for supplying power, and the DC-DC isolation module supplies power for positive voltage of the optical fiber receiver and supplies power for positive and negative voltage of the driving chip. The optical fiber receiver converts the control signal from an optical signal into an electric signal, and the driving chip amplifies the electric signal into a positive and negative bipolar driving signal to control the on-off of the solid-state switch group.
Referring to fig. 4, the set of solid state switches includes five solid state switches; each solid-state switch is connected in parallel in a fan-shaped symmetrical arrangement mode, the electric paths of the solid-state switches are the same while a high-frequency pulse magnetic field is generated, and the current equalizing effect is achieved.
Example 4:
the high-frequency pulse magnetic field induced cell magnetic perforation device comprises a high-voltage direct-current power supply, a pulse capacitor C, a switch driving device, a solid-state switch group 1, a magnetic field coil 2 and a discharge resistor R for protecting the stable work of a circuit;
the high-voltage direct-current power supply charges a pulse capacitor C;
the pulse capacitor C sends excitation pulse to the magnetic field coil 2 through the solid-state switch group 1;
the switch driving device controls the on-off of the solid-state switch group 1 to control the duration and the number of excitation pulses;
the switch driving device comprises an FPGA module, an electro-optical converter, a switch power supply and a turn-off driving module with negative pressure;
the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module;
the FPGA module sends a switch control electric signal to the electro-optical converter;
the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
the switching-off stable drive with the negative pressure comprises a DC-DC isolation module, an optical fiber receiver and a drive chip;
the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group 1 is controlled.
The solid-state switch group 1 comprises N solid-state switches; n is more than or equal to 2.
The N solid-state switches are arranged at intervals in the circumferential direction, wherein one ends of all the solid-state switches are connected to one point and recorded as a circle center O. The center O is connected with the capacitor C, and the other end of the solid-state switch is connected with the magnetic field coil.
The magnetic field coil 2 generates a magnetic field after receiving the excitation pulse, and the region of the magnetic field distribution is marked as a target action region;
an object to be treated is placed in the target action area;
and the object to be treated generates cell magnetic perforation under the action of a magnetic field.
And the FPGA module controls the on-off of the MOSFET switch group so as to control the duration and the number of the excitation pulses.
The equivalent circuit structure of the high-frequency pulse magnetic field induced cell magnetic perforation device is as follows:
recording that one end of a high-voltage direct-current power supply where a positive electrode is located is A, and one end of a negative electrode is B;
the A end is connected with the B end after being connected with the pulse capacitor C in series; recording the end of the pulse capacitor C which is not directly connected with the end A as D;
the A end is sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series and then grounded;
the A end is sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series and then connected with the B end;
the A end is connected with the D end after being sequentially connected with the solid-state switch group 1, the magnetic field coil 2 and the discharge resistor R in series.
The method for inducing the cell magnetic perforation device based on the high-frequency pulse magnetic field comprises the following steps:
1) building a high-frequency pulse magnetic field induced cell magnetic perforation device;
2) placing an object to be processed in a target action region of the magnetic field coil 2;
3) presetting pulse parameters;
4) the high-voltage direct-current power supply charges the energy storage capacitor C; the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module; the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
5) after the energy storage capacitor C is charged, the FPGA module generates a switch control electric signal based on a preset pulse parameter and transmits the switch control electric signal to the electro-optical converter;
6) the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
7) the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
8) the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group 1 is controlled;
the pulse capacitor C sends excitation pulse to the magnetic field coil 2 through the solid-state switch group 1;
9) the magnetic field coil 2 generates a magnetic field after receiving the excitation pulse, so as to induce the object to be processed in the target action region to generate magnetic cell perforation.
Example 5:
the method for inducing the cell magnetic perforation device based on the high-frequency pulse magnetic field comprises the following steps:
1) building a high-frequency pulse magnetic field induced cell magnetic perforation device;
2) placing an object to be processed in a target action region of the magnetic field coil 2;
3) presetting pulse parameters;
4) the high-voltage direct-current power supply charges the energy storage capacitor C; the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module; the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
5) after the energy storage capacitor C is charged, the FPGA module generates a switch control electric signal based on a preset pulse parameter and transmits the switch control electric signal to the electro-optical converter;
6) the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
7) the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
8) the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group 1 is controlled;
the pulse capacitor C sends excitation pulse to the magnetic field coil 2 through the solid-state switch group 1;
9) the magnetic field coil 2 generates a magnetic field after receiving the excitation pulse, so as to induce the object to be processed in the target action region to generate magnetic cell perforation.
Example 6:
a method for inducing a cell magnetic perforation device based on a high-frequency pulse magnetic field comprises the following steps:
1) carrying out adherence treatment on target cells in a pore plate;
2) placing the pore plate containing the cell solution in a target area of a magnetic field coil (2) of a magnetic perforation device;
3) presetting pulse parameters;
4) the high-voltage direct-current power supply charges the energy storage capacitor;
5) after the charging is finished, the FPGA module controls the on-off of the IGBT switch group based on preset pulse parameters to realize the output of pulses;
6) the output high-frequency pulse magnetic field causes the cells in the target area to be subjected to magnetic perforation.
Example 7:
referring to fig. 6 to 9, an experiment using a high-frequency pulsed magnetic field to induce cell magnetic perforation apparatus includes the following steps:
1) cell culture
The target cell used in the experiment is human skin cancer A375, which belongs to a malignant melanoma cell line.
1.1) cell passage
The supernatant from the T25 flask was decanted and washed twice with PBS, 1ml of 0.25% trypsin (25200056, Gibco) was added to the flask and placed in an incubator for digestion, 1ml of DMEM medium was added after 1 minute to stop digestion, DMEM medium was added after centrifugation for 5 minutes (800 rpm) and blown up again by a pipette until homogeneous, half of each was dispensed into a new T25 flask and 5ml of medium was replenished, and then incubation was continued in the incubator.
1.2) preparation of adherent cells
When the cells grow and are 80% confluent, the cells are digested and centrifuged (same cell passage step), a certain amount of DMEM medium is added, counting is carried out through a blood counting chamber, and finally the cell concentration is determined to be 2.5X 105And each/mL, placing the prepared cell suspension in a 48-well plate for 24 hours, and performing pulse treatment after the cells are completely attached to the wall.
2) Establishment of experimental platform
The schematic diagram of the experimental platform device constructed in this embodiment is shown in fig. 1, and a375 cell suspension prepared in advance is filled into a 48-well plate (the diameter of each well is 1cm), and the plate is kept still for 24 hours to wait for the adhesion of cells. After the cells are attached to the wall, the 48-hole plate is placed at the output end of a self-made high-frequency magnetic field pulse generator in a laboratory, and the holes containing the cells are correspondingly placed right above the magnetic field generating coil. Meanwhile, the high-voltage probe is connected to two ends of a resistor connected with the coil in parallel, and finally, the voltage waveforms at the two ends of the resistor are acquired through an oscilloscope. Because the resistance is in parallel relation with the coil generating the magnetic field, the voltage waveform obtained by the test is considered to be the voltage waveform received by the magnetic field generating coil.
The schematic structural diagram of the pulse generator device used in this embodiment is shown in fig. 2, programming is performed through a PC end of a computer, a program is burned into a field programmable gate array module, and signal output of an FPGA is transmitted to an IGBT switch through an optical fiber to control on and off of the IGBT, so that control over output pulse parameters is realized. The nanosecond pulse generator body adopts a traditional RC charge-discharge circuit structure, namely, a capacitor is charged through a high-voltage direct-current power supply, and then the action of an IGBT switch is controlled through an output signal of an FPGA, so that the duration time and the action number of pulse voltage at two ends of a load are controlled.
3) A375 cell adherence Effect assay
The same volume of cells as the concentration of the cells used in the experiment was placed in a 48-well plate, the cells and the plate were allowed to stand together in an incubator for 24 hours, and after 24 hours, the cells were observed using a microscope, at which time the cells were fusiform. And then, using a PBS solution which is prepared in advance in a laboratory and has no influence on the cell state to slightly wash the cells, and placing the washed cells under an optical microscope again for observation to obtain the cells which are still fusiform, wherein the cell number is basically consistent with that of the cells before washing, which indicates that the cells can be completely attached to the wall on a pore plate after 24 hours. Because only living cells can adhere to the wall, the cells adhere to the wall and dead cells can be screened out when the subsequent experiment treatment is facilitated, and the cells after adhering to the wall can approach the magnetic field generating coil to a greater extent and are beneficial to obtaining better experiment effect under the condition of smaller magnetic field amplitude.
4) High-frequency pulse magnetic field treatment scheme
The pulse parameters adopted in the adherent cell experiment in the step are shown in table 1, and the influence mechanism of the change of the pulse frequency on the cells is relatively complex, and the purpose of the experiment is to explore the influence of the pulsed magnetic field on the cell perforation effect under the high-frequency condition, so that the pulse frequency in the fixed string in the experiment is 100kHz, and the pulse frequency outside the fixed string is 1 Hz. And then 5 parameter values with different levels are respectively set for two variables of the magnetic field amplitude average value (B) and the pulse number (N), and the parameter values are determined through exploration of early-stage pre-experiments and respectively generate weak-to-strong influence on cells. The present embodiment first sets an intermediate value for the parameters, i.e., B is 310.3mT and N is 2 × 104And (4) respectively. When B is changed, N is fixed to 2X 104A plurality of; when changing N, then B is fixed at 310.3 mT.
TABLE 1 Experimental parameter table for high-frequency pulse magnetic field
Table 1 Experimental parameters of nsPMFs
Figure BDA0002926229560000101
The method of step 1 is used for obtaining cells attached to the wall in a pore plate, wherein the attached cells in each experiment are divided into an experiment group which is added with a magnetic field for treatment and a blank group which is respectively contrasted with different experiment groups and is not added with any treatment. The 48-hole plate which is paved in advance is placed right above the magnetic field generator, and then a pulse magnetic field with specific parameters is applied for treatment. And after the pulse magnetic field treatment is finished, placing the orifice plate in an incubator for 48 hours. After 48 hours of incubation, cck-8 reagent was added, and after 1.5 hours of incubation, absorbance was measured by a microplate reader.
5) PI staining method for detecting cell membrane permeability
5.1) PI reagent staining method
In this step, trypsin without EDTA was used to digest the cells. After the pulsed magnetic field treatment, the treated cells and the well plate were directly placed in an incubator and incubated for 3 hours. After 3 hours the cells were digested from the 48-well plates with trypsin without ethylenediaminetetraacetic acid, centrifuged 3 times to remove the medium from the cell solution, added PBS buffer and finally brought to a volume of 200 μ L. mu.L of a mixed solution of PBS and Propidium Iodide (PI) (20: 1) was added in the dark and incubation was continued for 10 minutes, and finally detection was performed by flow cytometry in the dark.
PI is a macromolecular nucleic acid dye, and when the cell membrane has an intact morphology, PI cannot penetrate through the cell membrane to enter the interior of the cell and be bound with the nucleus. When the outer cell membrane is perforated under the action of a pulse magnetic field, PI molecules can penetrate through the cell membrane through micropores in the membrane, enter the interior of the cell and are combined with the cell nucleus. Therefore, the PI staining method can accurately reflect the change of the membrane permeability after the cells are subjected to magnetic perforation, and finally qualitatively characterize the strength of the magnetic perforation effect through the proportion of PI positive cells.
5.2) Effect of pulse parameter variation on the proportion of PI-Positive cells
FIGS. 6 and 7 are histograms of the percentage of PI-positive cells under different pulse parameters. As shown in the figure, the PI positive ratio of the cells is low and no significant difference exists in the control group (p >0.05) because no pulse magnetic field is applied, which indicates that perforation does not occur basically and the cell membrane is in an intact state.
When the magnetic field amplitude was changed, the PI positive ratio of the cells increased from 1.8% of 103.4mT to 36.7% of 517.1mT under the action of nsPMFs, which was a very significant difference compared to the blank control group (p <0.01, indicating the magnitude of the difference in the statistical analysis), indicating that the a375 melanoma cells used in the experiment were indeed perforated after the treatment with the application of the pulsed magnetic field.
When the number of pulses is changed, the proportion of positive PI of the cells under the action of nsPMFs is 1X 1041.1% rise to 3X 10 under one pulse438.9% under each pulse. And has a very significant difference (p) compared with a blank control group<0.01). The above experimental results all show that the experimental institute is added with the pulsed magnetic field for treatmentThe a375 melanoma cells used did undergo perforation.
Therefore, based on the above experimental results, it can be found that the melanoma cells of the target cell a375 are capable of magnetic perforation under the pulse parameters used in the experiment, and the degree of perforation is enhanced with the enhancement of the pulse parameters.
6) PI staining method for detecting proportion of cells generating perforation
6.1) PI reagent staining method
In this step, trypsin without EDTA was used to digest the cells. After the pulsed magnetic field treatment, the treated cells and the well plate were directly placed in an incubator and incubated for 30 minutes. After 30 minutes, the cells were gently rinsed 2 times with pre-prepared PBS to ensure that the cell surface media was washed clean. Then 200. mu.L of a mixed solution (100: 1) of PBS and Propidium Iodide (PI) was added in the dark and incubation was continued for 10 minutes, and finally detection was performed by fluorescence microscopy in the dark.
In step 5, the change of the membrane permeability after the magnetic perforation of the cells can be accurately reflected by using a PI staining method. The ratio of stained cells to total cells in the field of view observed by statistical fluorescence microscopy can be used to determine the ratio of cells undergoing magnetic perforation in relation to different pulse parameters.
6.2) Effect of pulse parameter variation on the proportion of cells undergoing perforation
FIGS. 8 and 9 are bar graphs of the percentage of stained cells generated by different pulse parameters. As shown in fig. 8, in the control group, since no pulsed magnetic field was applied, staining of the cells was not observed, indicating that perforation did not occur substantially and the cell membrane was intact.
When the magnetic field amplitude is changed, the proportion of stained cells is increased from 1.7% of 103.4mT to 20.2% of 517.1mT under the action of nsPMFs, and the cells have a very significant difference (p <0.01) compared with a blank control group, which indicates that the A375 melanoma cells used in the experiment are actually perforated after the pulsed magnetic field is added for treatment, and the number of perforated cells is increased along with the increase of the pulse magnetic field amplitude.
When the number of pulses is changed, the proportion of positive PI of the cells under the action of nsPMFs is 1X 1042.0% rise to 3X 10 under one pulse410.9% under each pulse. And when the number of pulses is 2 x 104And 2.5X 104At all times, the experimental group had significant differences (p) from the blank control group<0.05); when the number of pulses is 3 x 104At all times, there was a very significant difference (. about.p) between the experimental group and the blank control group<0.01). The above experimental results all show that the a375 melanoma cells used in the experiment are actually perforated after the pulsed magnetic field is applied for treatment, and the number of the perforated cells increases with the increase of the amplitude of the pulsed magnetic field. Therefore, based on the above experimental results, it was found that the melanoma cells of the target cell a375 were able to be magnetically perforated under the pulse parameters used in the experiment, and the proportion of the cells where perforation occurred increased with the increase of the pulse parameters.

Claims (7)

1. The high-frequency pulse magnetic field induced cell magnetic perforation device is characterized by comprising a high-voltage direct-current power supply, a pulse capacitor C, a switch driving device, the solid-state switch group (1) and a magnetic field coil (2).
The high-voltage direct-current power supply charges a pulse capacitor C;
the pulse capacitor C sends excitation pulses to the magnetic field coil (2) through the solid-state switch group (1);
the switch driving device controls the on-off of the solid-state switch group (1) to control the duration and the number of excitation pulses;
the magnetic field coil (2) generates a magnetic field after receiving the excitation pulse, and the region of the magnetic field distribution is marked as a target action region;
an object to be treated is placed in the target action area;
and the object to be treated generates cell magnetic perforation under the action of a magnetic field.
And the FPGA module controls the on-off of the MOSFET switch group so as to control the duration and the number of the excitation pulses.
2. The apparatus for inducing magnetic perforation of cells by high-frequency pulsed magnetic field according to claim 1, wherein: and the discharge resistor R is used for protecting the stable operation of the circuit.
3. The apparatus for inducing magnetic perforation of cells by high-frequency pulsed magnetic field according to claim 2, wherein: the equivalent circuit structure of the high-frequency pulse magnetic field induced cell magnetic perforation device is as follows:
recording that one end of a high-voltage direct-current power supply where a positive electrode is located is A, and one end of a negative electrode is B;
the A end is connected with the B end after being connected with the pulse capacitor C in series; recording the end of the pulse capacitor C which is not directly connected with the end A as D;
the A end is sequentially connected with the solid-state switch group (1), the magnetic field coil (2) and the discharge resistor R in series and then grounded;
the A end is sequentially connected with the solid-state switch group (1), the magnetic field coil (2) and the discharge resistor R in series and then connected with the B end;
the A end is sequentially connected with the solid-state switch group (1), the magnetic field coil (2) and the discharge resistor R in series and then connected with the D end.
4. The apparatus for inducing magnetic perforation of cells by high-frequency pulsed magnetic field according to claim 1, wherein: the switch driving device comprises an FPGA module, an electro-optical converter, a switch power supply and a turn-off driving module with negative pressure;
the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module;
the FPGA module sends a switch control electric signal to the electro-optical converter;
the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
the switching-off stable drive with the negative pressure comprises a DC-DC isolation module, an optical fiber receiver and a drive chip;
the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group (1) is controlled.
5. The apparatus for inducing magnetic perforation of cells by high-frequency pulsed magnetic field according to claim 1, wherein: the solid-state switch group (1) comprises N solid-state switches; n is more than or equal to 2.
6. The apparatus for inducing magnetic perforation of cells by high-frequency pulsed magnetic field according to claim 1, wherein: the N solid-state switches are arranged at equal intervals in the circumferential direction, wherein one ends of all the solid-state switches are connected to one point and recorded as a circle center O.
7. The method for inducing the magnetic cell perforation by the high-frequency pulse magnetic field according to any one of claims 1 to 6, comprising the steps of:
1) building a high-frequency pulse magnetic field induced cell magnetic perforation device;
2) placing an object to be processed in a target action area of the magnetic field coil (2);
3) presetting pulse parameters;
4) the high-voltage direct-current power supply charges the energy storage capacitor C; the switch power supply supplies power to a DC-DC isolation module with a negative pressure turn-off driving module; the DC-DC isolation module supplies power to the optical fiber receiver and the driving chip;
5) after the energy storage capacitor C is charged, the FPGA module generates a switch control electric signal based on a preset pulse parameter and transmits the switch control electric signal to the electro-optical converter;
6) the electro-optical converter converts the switch control electrical signal into a switch control optical signal and sends the switch control optical signal to the switch power supply and the turn-off driving module with negative pressure;
7) the optical fiber receiver receives the switch control optical signal and restores the switch control optical signal into a switch control electric signal; the optical fiber receiver transmits the switch control electric signal to the driving chip;
8) the driving chip generates positive and negative bipolar driving signals based on the switch control electric signals, so that the on-off of the solid-state switch group (1) is controlled;
the pulse capacitor C sends excitation pulses to the magnetic field coil (2) through the solid-state switch group (1);
9) the magnetic field coil (2) generates a magnetic field after receiving the excitation pulse, so that the object to be treated in the target action region is induced to generate magnetic cell perforation.
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