A cell membrane electric shock freezing experimental method and device

Abstract

The present invention relates to the field of medical experimental equipment, and in particular to a cell membrane electric shock freezing experimental method and device. Existing ultrafast recording cameras are unable to take photos of the actual effect of nanosecond pulse electric fields destroying cell membrane microstructures in real-time nanosecond pulse applications, and lack direct morphological evidence. The present invention provides a cell membrane electric shock freezing experimental method and device, the experimental method comprising the following steps in sequence: (1) preparing agarose gel solution and cooling it to 35-40°C; (2) adding cells cultured to the logarithmic growth phase to the agarose gel solution, and then shaking it until the agarose solution solidifies into a solid jelly; (3) the solid jelly passes through a parallel electric field in a free fall manner and then falls into liquid nitrogen for freezing and solidification. The present invention can capture the instantaneous perforation phenomenon of the cell membrane when the nanosecond pulse electric field is applied, and quickly freezes and solidifies the perforated cell membrane, providing reliable morphological evidence and facilitating subsequent research.

Description

Technical Field

[0001] The invention relates to the field of medical experimental equipment, in particular to a cell membrane electric shock freezing experimental method and device. Background Art

[0002] Nanosecond pulsed electric field (nsPEF) is a non-thermal, non-ionizing, non-radiative physical method that can use strong electric fields to ablate tumor cells. Nanosecond pulsed electric field ablation has the characteristics of high output power but no obvious heat accumulation. Existing studies have shown that nanosecond pulsed electric fields can affect cell function, but the exact molecular mechanism is still unclear. Some researchers have found that nanosecond pulsed electric fields can expand traditional electroporation to form nanopores. Further studies have also found that nanosecond pulsed electric fields can destroy microdomains on cell membranes. Existing studies have provided indirect evidence of increased permeability of cell membranes marked by fluorescent dyes or analogs, but current ultrafast recording cameras are unable to take photos of the actual effects of nanosecond pulsed electric fields in real-time nanosecond pulse applications. Therefore, there is no reliable direct morphological evidence to prove the formation of membrane nanopores. Summary of the invention

[0003] The technical problems to be solved and the technical tasks proposed by the present invention are to overcome the existing lack of effective direct morphological evidence for the formation of membrane nanopores by nanosecond pulse electric field (nsPEF) destroying microstructure domains on the cell membrane, and the inability of existing ultrafast recording cameras to take photos of the actual effects of nanosecond pulse electric fields in real-time nanosecond pulse applications. The present invention provides a cell membrane electric shock freezing experimental method and device, which can capture the instantaneous perforation phenomenon caused by the application of nanosecond pulse electric fields on cell membranes, and quickly freeze and solidify the morphological changes of cell membranes that have undergone instantaneous perforation, so as to provide direct and reliable morphological evidence, and facilitate subsequent research on dynamic membrane floating and nanopore perforation of cell membranes.

[0004] The technical solution adopted by the present invention to solve the technical problem is: a cell membrane electric shock freezing experimental method, characterized in that the experimental method comprises the following steps performed in sequence:

[0005] (1) Prepare agarose gel solution and cool it to 35-40°C;

[0006] (2) Add cells cultured in the logarithmic growth phase to the agarose gel solution, then shake well and wait for the agarose solution to solidify into a solid jelly;

[0007] (3) The solid jelly falls freely through a parallel electric field and then falls into liquid nitrogen where it freezes and solidifies.

[0008] The invention puts cells into agarose gel solution to form solid jelly, then makes the solid jelly do free fall motion through a parallel electric field to be subjected to flash electric shock, and then drops into liquid nitrogen in a flash freezing manner after nanosecond pulse electric shock is implemented to achieve transient freezing solidification and preservation, and provides direct morphological evidence within a millisecond level time period after the pulse occurs, so as to show the instantaneous change of the cell membrane after the nanosecond pulse electric shock of the cell, and facilitates the study of membrane images and cell medium properties; after the electric shock is completed, the solid jelly instantly drops into liquid nitrogen, and the cells treated with the nanosecond pulse are immediately fixed before ice crystals are formed, so as to avoid ice crystal effect and damage of the cell membrane.

[0009] As a further improvement and supplement to the above technical solution, the present invention adopts the following technical measures: the time taken by the solid jelly from the beginning of falling to the completion of electric shock is 247ms, the parallel electric field is a nanosecond pulse electric field, and then enters liquid nitrogen within 2ms. The time taken by the solid jelly from the beginning of falling to the completion of electric shock is 247ms, the parallel electric field is a nanosecond pulse electric field, and then enters liquid nitrogen within 2ms. The solid jelly blocks the cells at a specific time point of 247ms after the implementation of nanosecond pulse electric shock in a flash freezing manner, and then falls into liquid nitrogen within 2ms to achieve transient freezing and solidification preservation, providing direct morphological evidence within the millisecond level time period after the pulse occurs, so as to show the instantaneous changes of the cell membrane after the nanosecond pulse electric shock, and facilitate the study of membrane images and cell medium properties; after the electric shock is completed, the solid jelly falls into liquid nitrogen within 2ms, which can immediately fix the cells treated with nanosecond pulses before ice crystals are formed, avoiding ice crystal effects and damage to the cell membrane.

[0010] The nanosecond pulse electric field is a rectangular nanosecond pulse, the duration of the nanosecond pulse is 300ns, the field strength is 8kv / cm, and the fast pulse rising edge time is ≤20ns. The cell membrane is electroshocked with millisecond-level electric pulses to effectively destroy the microstructure domains on the cell membrane.

[0011] The agarose gel solution is a 0.8% agarose gel solution prepared by mixing agarose and pure water and heating in a microwave oven. The 0.8% agarose gel solution has a better effect in the electric shock freezing experiment and is convenient for studying membrane images and cell media.

[0012] A cell membrane electric shock freezing experimental device, comprising a liquid nitrogen storage container, a base, an electric shock mechanism, a free fall mechanism and a detachable cell clamp, wherein the base is mounted above the liquid nitrogen storage container, the cell clamp comprises an insulating annular ring gasket and two copper sheets, the two copper sheets can clamp the gasket to form a closed cavity; the electric shock mechanism comprises a nanosecond pulse generator and positive and negative electrode plates connected to the wires thereof, the positive and negative electrode plates are vertically arranged on the base, and the base is provided with a gap between the positive and negative electrode plates; the free fall mechanism The structure includes a guide rail arranged on a base and a slider arranged thereon and capable of free-falling motion. The slider is located above the neutral position and a clamping structure for clamping a cell clamp is arranged at the bottom thereof. The clamping structure can make the cell clamp free-fall in a state where the two copper sheets are kept parallel to the positive and negative electrodes. When the cell clamp passes between the positive and negative electrodes with the slider in free-falling motion, the positive and negative electrodes discharge and shock the cell clamp. When the slider falls to the lowest position, the cell clamp is loosened from the clamping structure and passes through the neutral position and falls into a liquid nitrogen storage container. The present invention can make agarose gel solid jelly containing cells fall freely, and apply electric shock of nanosecond pulse electric field to it during the fall, and then quickly drop it into liquid nitrogen for freezing and solidification. Through the combination of flash electric shock and flash freezing, direct morphological evidence is provided within a millisecond time period after the pulse occurs, showing the instantaneous changes of the cell membrane after the nanosecond pulse electric shock to the cell. During operation, the jelly can be placed in the middle of the annular ring pad and then clamped with two copper sheets and placed in the clamping structure at the bottom of the slider. Then the slider performs free fall along the guide rail. During the falling process, the copper sheets on both sides of the cell clamp remain parallel to the positive and negative electrodes. When the jelly passes through the area between the positive and negative electrodes, the nanosecond pulse generator works and applies a pulse electric field shock to the jelly. When the slider falls to the lowest position, the cell clamp is loosened from the clamping structure and passes through the neutral position to fall into the liquid nitrogen previously filled in the liquid nitrogen storage container. The cell clamp opens after colliding with the liquid nitrogen surface, and the two copper sheets separate. The jelly is separated from the annular gasket and immersed in the liquid nitrogen for freezing and solidification to achieve freezing, freezing the instantaneous perforation of the cell membrane caused by the nanosecond pulse electric field. In addition, the nanosecond pulse generator is a prior art, and the present invention uses a Blumlein type nanosecond pulse generator of the prior art.

[0013] The guide rails are two vertical guide rods arranged on the base, and the slider has holes at both ends thereof and is sleeved on the two guide rods. The guide rail structure is simple, and two vertical guide rods can be used. The slider is sleeved on the guide rods through the holes at both ends thereof, ensuring that the slider slides down along the guide rods to perform free fall motion, and keeping the two copper sheets of the cell clamp held on the slider parallel to the positive and negative electrode plates.

[0014] The clamping structure is a clamping groove that is open downward and can be tightly matched with the cell clamp at the bottom of the slider. A collision seat that matches the slider is set between the positive and negative electrode plates on the base. When the slider falls to the lowest position, it hits the collision seat to make the cell clamp loosen from the clamping groove and fall. Since the clamping groove is tightly matched with the cell clamp, when the slider falls to the lowest position, it hits the collision seat to generate vibration, so that the originally tightly matched cell clamp is loosened from the clamping groove and falls into the liquid nitrogen storage container.

[0015] The neutral position is a strip hole set through on the base, the positive and negative electrode plates are separated on both sides of the length direction of the strip hole and extend along the length direction of the strip hole, the collision seat is provided with a through hole matching the strip hole, the top surface of the slider and the bottom surface of the collision seat are provided with matching collision surfaces, when the slider falls to the lowest position, the slider hits the collision seat to make the cell clamp fall off, and passes through the through hole and the strip hole from top to bottom and falls into the liquid nitrogen storage container. The base directly opens a through strip opening to form a neutral position suitable for the cell clamp to fall through; the positive and negative electrode plates are separated on both sides of the length direction of the strip hole and extend along its length direction, forming a parallel electric field area matching the length of the strip hole, which is convenient for discharging the cell clamp passing through and the cell jelly set therein; when the cell clamp falls, it can pass through the through hole on the collision seat and the strip hole on the base in turn, and then fall into the liquid nitrogen; the slider and the collision seat cooperate through the collision surfaces set at the corresponding contact parts, and there is enough area for collision to produce appropriate vibration, so as to ensure that the cell clamp is loosened and falls off.

[0016] The collision surface on the collision seat and the slider is a plane of matching size, and the clamping groove is composed of two parallel clamping plates fixed on the bottom surface of the slider, and the two clamping plates protrude downward from the bottom surface of the slider. The collision surface is a plane of matching size set on the collision seat and the slider, and the two plane structures are simple and easy to process. When the two planes on the slider and the collision seat are in contact, the slider falls to the lowest position; the two clamping plates protrude downward from the bottom surface of the slider, and when the slider and the collision seat collide, the two protruding clamping plates can have active space for displacement to shake the cell clamp off.

[0017] The present invention provides a cell membrane electric shock freezing experimental method and device, which can capture the instantaneous perforation phenomenon when a nanosecond pulse electric field is applied to the cell membrane, and quickly freeze and solidify the morphological changes of the cell membrane that undergoes instantaneous perforation, so as to provide direct and reliable morphological evidence, thereby facilitating subsequent cell membrane dynamic membrane floating and nanopore perforation research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic diagram of the overall structure of the present invention.

[0019] Figure 2 : Figure 1 AA section view.

[0020] Figure 3 : Figure 1 BB section view.

[0021] Figure 4 : Figure 1 Working principle of the device Figure 1 (Slider in upper position).

[0022] Figure 5 : Figure 1 Working principle of the device Figure 2 (Slider in lowest position).

[0023] In the figure: 1. liquid nitrogen storage device, 2. base, 3. ring gasket, 4. copper sheet, 5. wire, 6. positive electrode plate, 7. negative electrode plate, 8. slider, 9. guide rod, 10. clamping groove, 11. collision seat, 12. strip hole, 13. through hole, 14. clamping plate. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods.

[0025] like Figures 1 to 5As shown, a cell membrane electric shock freezing experimental device includes a liquid nitrogen storage container 1, a base 2, an electric shock mechanism and a free fall mechanism and a detachable cell clamp, wherein the base 2 is mounted above the liquid nitrogen storage container 1, and the cell clamp includes an insulating annular ring gasket 3 and two copper sheets 4, and the two copper sheets 4 can clamp the gasket 3 to form a closed cavity; the electric shock mechanism includes a nanosecond pulse generator and a positive electrode plate 6 and a negative electrode plate 7 connected to the wire 5 and arranged opposite to each other, the positive and negative electrode plates are vertically arranged on the base 2, and the base 2 is provided with a gap between the positive electrode plate 6 and the negative electrode plate 7; the free fall mechanism The invention comprises a guide rail arranged on a base 2 and a slider 8 arranged thereon and capable of free-falling motion. The guide rail is two vertical guide rods 9 arranged on the base 2. The two ends of the slider 8 have openings sleeved on the two guide rods 9. The slider 8 is located above the neutral position and a clamping structure for clamping the cell clamp is arranged at the bottom thereof. The cell clamp can be free-falling in a state where the two copper sheets 4 are kept parallel to the positive electrode plate 6 and the negative electrode plate 7. When the cell clamp passes between the positive electrode plate 6 and the negative electrode plate 7 while the slider 8 is free-falling, the nanosecond pulse generator passes through the positive electrode plate 6 and the negative electrode plate 7. 7 discharges electricity to shock the cell clamp. When the slider 8 falls to the lowest position, the cell clamp is loosened from the clamping structure and passes through the gap to fall into the liquid nitrogen pre-filled in the liquid nitrogen storage container 1; the clamping structure is a clamping groove 10 that is set at the bottom of the slider 8 and is open downward and can be tightly matched with the cell clamp. The clamping groove 10 is composed of two parallel clamping plates 14 fixed on the bottom surface of the slider 8, and the two clamping plates 14 protrude downward from the bottom surface of the slider 8; a collision seat 11 that matches the slider 8 is set between the positive electrode plate 6 and the negative electrode plate 7 on the base 2. When the slider 8 falls to the lowest position, it hits the collision seat 11 to make the cell clamp come out of the clamping groove 10 is loosened and falls; the neutral position is a strip hole 12 which is set through on the base 2, the positive electrode plate 6 and the negative electrode plate 7 are separated on both sides of the length direction of the strip hole 12, and extend along the length direction of the strip hole 12, the collision seat 11 is provided with a through hole 13 which matches the strip hole 12, the top surface of the slider 8 and the bottom surface of the collision seat 11 are respectively provided with matching collision surfaces, and the collision surfaces on the collision seat 11 and the slider 8 are planes of matching sizes; when the slider 8 falls to the lowest position, the slider 8 hits the collision seat 11 to make the cell clamp fall off, and passes through the through hole 13 and the strip hole 12 from top to bottom and falls into the liquid nitrogen storage container 1.

[0026] When working, the jelly is placed in the middle of the annular ring pad and then clamped by two copper sheets into the clamping structure at the bottom of the slider. Then the slider performs free fall along the guide rail. In this embodiment, the preset height h of the slider is 0.3m. According to the physical formula h=1 / 2 gt 2It can be calculated that the free fall is 0.247s, the nanosecond pulse generator discharges, and a parallel electric field is formed between the positive and negative electrode plates, which shocks the cell clamp. During the fall, the two copper sheets of the cell clamp remain parallel to the positive and negative electrode plates; when the slider falls to the lowest position, the cell clamp loosens from the clamping structure and passes through the gap and falls into the liquid nitrogen pre-filled in the liquid nitrogen storage container. The height difference between the lower edge of the positive and negative electrode plates and the liquid nitrogen level is 10mm. After the cell clamp hits the liquid nitrogen surface, it opens, and the two copper sheets separate. The jelly is separated from the annular gasket and immersed in liquid nitrogen for freezing and solidification. The instantaneous perforation changes of the cell membrane caused by the nanosecond pulse electric field are captured, and the cells in the solid jelly will be dispersed into the liquid nitrogen. For comparison, the aforementioned liquid nitrogen storage container can be replaced with a culture dish, and the cells treated with the aforementioned electric shock are dropped into the culture dish. The cells treated with the nanosecond pulse can be cultured in vitro, and the cell cycle, apoptosis, survival and growth can be analyzed, and compared with the cells frozen after the aforementioned electric shock.

[0027] A cell membrane electric shock freezing experimental method, the experimental method comprising the following steps performed in sequence:

[0028] (1) Prepare agarose gel solution and cool it to 35-40°C; the agarose gel solution is a 0.8% agarose gel solution prepared by mixing agarose and pure water and heating it in a microwave oven; in this embodiment, 0.4 g of agarose is weighed and placed in a 200 ml conical flask, 50 ml of pure water is added, and the flask is placed in a microwave oven and heated until the agarose is completely melted, then taken out and shaken to obtain a 0.8% agarose gel solution, which is then placed at room temperature and cooled to 37°C by touch;

[0029] (2) Add cells cultured in the logarithmic growth phase to the agarose gel solution, then shake it well. Wait for the agarose solution to solidify into a solid jelly. Then, fill the solid jelly into the middle of the ring pad between the two copper plates of the cell clamp, and then put it into the gap formed by the two clamps at the bottom of the slider;

[0030] (3) The solid jelly passes through a parallel electric field in a free fall manner and then falls into liquid nitrogen to be frozen and solidified. The time taken for the solid jelly from the beginning of falling to the completion of electric shock is 247 ms. The parallel electric field is a nanosecond pulse electric field, and then enters the liquid nitrogen within 2 ms; the nanosecond pulse electric field is a rectangular nanosecond pulse, the duration of the nanosecond pulse is 300 ns, the field strength is 8 kV / cm, and the fast pulse rising edge time is ≤20 ns. During the above-mentioned free fall process, the slider falls along the guide rod until it hits the collision seat at the lowest position, and the cell clamp breaks away from the slider and falls into the liquid nitrogen. The pulse shape can be recorded and monitored by an oscilloscope.

Claims

1. A cell membrane electric shock freezing experimental device, comprising a liquid nitrogen storage container (1), a base (2), an electric shock mechanism and a free fall mechanism and a detachable cell clamp, wherein the base (2) is mounted above the liquid nitrogen storage container (1), the cell clamp comprises an insulating annular ring gasket (3) and two copper sheets (4), the two copper sheets (4) can clamp the gasket (3) to form a closed cavity; the electric shock mechanism comprises a nanosecond pulse generator and positive and negative electrode plates (6, 7) connected to the wire (5) and arranged opposite to each other, the positive and negative electrode plates are vertically arranged on the base (2), and the base (2) is provided with a gap between the positive and negative electrode plates (6, 7); The free fall mechanism comprises a guide rail arranged on a base (2) and a slider (8) arranged thereon and capable of free fall movement. The slider (8) is located above the neutral position and a clamping structure for clamping a cell clamp is arranged at the bottom thereof. The clamping structure can make the cell clamp free fall in a state where two copper sheets (4) are kept parallel to the positive and negative electrode plates (6, 7). When the cell clamp passes between the positive and negative electrode plates (6, 7) during free fall movement along with the slider (8), the positive and negative electrode plates (6, 7) discharge and shock the cell clamp. When the slider (8) falls to the lowest position, the cell clamp is loosened from the clamping structure, passes through the neutral position and falls into the liquid nitrogen storage container (1).

2. The cell membrane electric shock freezing experimental device according to claim 1 is characterized in that The guide rails are two vertical guide rods (9) arranged on the base (2), and the sliding block (8) has holes at both ends thereof and is sleeved on the two guide rods (9).

3. The cell membrane electric shock freezing experimental device according to claim 1 is characterized in that The clamping structure is a clamping groove (10) which is arranged at the bottom of the slider (8) and is open downward and can be tightly matched with the cell clamp. A collision seat (11) matching the slider (8) is arranged between the positive and negative electrode plates (6, 7) on the base (2). When the slider (8) falls to the lowest position, it hits the collision seat (11) to make the cell clamp loosen and fall from the clamping groove (10).

4. The cell membrane electric shock freezing experimental device according to claim 3 is characterized in that The neutral position is a strip hole (12) provided through the base (2); the positive and negative electrode plates (6, 7) are provided on both sides of the strip hole (12) in the length direction and extend along the length direction of the strip hole (12); the collision seat (11) is provided with a through hole (13) matching the strip hole (12); the top surface of the slider (8) and the bottom surface of the collision seat (11) are provided with matching collision surfaces; when the slider (8) falls to the lowest position, the slider (8) hits the collision seat (11) to cause the cell clamp to fall off, and then passes through the through hole (13) and the strip hole (12) from top to bottom and falls into the liquid nitrogen storage container (1).

5. The cell membrane electric shock freezing experimental device according to claim 4 is characterized in that The collision seat (11) and the collision surface on the slider (8) are planes of matching size, and the clamping groove (10) is composed of two parallel clamping plates (14) fixed on the bottom surface of the slider (8), and the two clamping plates (14) protrude downward from the bottom surface of the slider (8).

6. A cell membrane electric shock freezing experimental method, characterized in that The experimental method uses the cell membrane electric freezing experimental device described in any one of claims 1 to 5 to perform the experiment, and the experimental method includes the following steps performed in sequence: (1) preparing agarose gel solution and cooling it to 35-40° C.; the agarose gel solution is a 0.8% agarose gel solution prepared by mixing agarose and pure water and heating it in a microwave oven; (2) adding cells cultured in the logarithmic growth phase to the agarose gel solution, then shaking until the agarose solution solidifies into a solid jelly; (3) The solid jelly falls freely through a parallel electric field and then falls into liquid nitrogen and freezes and solidifies; The time taken for the solid jelly from the beginning of falling to the completion of electric shock is 247ms, the parallel electric field is a nanosecond pulse electric field, and then it enters the liquid nitrogen within 2ms; The nanosecond pulse electric field is a rectangular nanosecond pulse, the duration of the nanosecond pulse is 300ns, the field strength is 8kv / cm, and the fast pulse rising edge time is ≤20ns.

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