A pressurizable flow electroporation device

Abstract

The pressurized flow electrode tube device of the present invention is based on the principle of a water pressure well and provides an electrotransfection device that can automatically fill liquid and pressurize it through a movable piston, inhibit the generation of bubbles in the cell fluid during electroporation, and effectively remove dead cells and other paste-like substances accumulated and attached to the electrode and the tube body.

Description

Technical Field

[0001] The present invention relates to the field of electroporation, and to a pressurizable flow electroporation device. More specifically, the present invention relates to a flow electroporation device which suppresses the generation of bubbles during the electroporation process by piston pressurization. Background Art

[0002] The cell membrane is a thin film surrounding the cell, and is a permeable barrier for the selective exchange of substances between the cell and the outside world. The cell membrane makes the cell an independent life unit and has a relatively stable internal environment. Some substances in the surrounding environment can pass through the cell membrane, while others cannot. Cells can absorb nutrients from the surrounding environment and excrete metabolic products through the cell membrane, so that the transport of substances reaches a balanced state. Therefore, the basic function of the cell membrane is to maintain the relative stability of the intracellular microenvironment and selectively exchange substances with the external environment.

[0003] Research has found that if a certain intensity of electrical stimulation is applied to cells for a period of time, some micropores can be induced on the cell membrane, which can enhance the permeability of the cells. The so-called cell electroporation refers to the biophysical process in which transient micropores are formed on the lipid bilayer of the cell membrane under the action of an external pulsed electric field. Electrotransfection is a technology that uses electroporation technology to introduce exogenous biomacromolecules, such as DNA, RNA or protein, into cells. When the cell membrane is electroporated, its permeability and membrane conductance will increase instantaneously, allowing hydrophilic molecules, DNA, proteins, virus particles, drug particles and other molecules that cannot pass through the cell membrane under normal circumstances to enter the cell through the micropores. After the electrical stimulation is removed in a short period of time, the micropores on the cell membrane disappear, and the cell membrane becomes a selective permeable barrier again.

[0004] Compared with traditional chemical transfection and viral transfection, electrotransfection has broad application prospects in biophysics, molecular biology, clinical medicine and other fields due to its advantages such as no chemical pollution, no permanent damage to cells and high efficiency.

[0005] Although the mechanism of electrofection is not completely clear, it is well known in the art that electrofection of cells involves the movement of the lipid bilayer of the cell membrane, resulting in the formation of temporary micropores in the membrane, allowing exogenous molecules to enter the cell through the micropores.

[0006] In the prior art, there are three main methods for completing the process of cell electrotransfection: placing cells between a pair of parallel electrodes separated by several millimeters to several centimeters, so that the cells are electrically stimulated in the electric field between the electrodes to achieve the purpose of electrotransfection. For example, U.S. Patent No. 5,389,069.

[0007] Use micro needle electrodes to penetrate into tissues or cell fluids to shock cells, thereby achieving the purpose of electrotransfection. For example, US Pat. No. 5,389,069.

[0008] A chamber is placed between a pair of parallel electrodes so that a suspension of cells is subjected to electric shock while flowing in the chamber. For example, US Pat. No. 6,773,669.

[0009] Chinese patent CN201010242144 discloses a flow electrotransfection device and system, which includes: a flow electrotransfection device, including: a substrate, and electrodes made on the substrate, the electrodes are placed in parallel and in pairs, and each pair of electrodes includes an anode and a cathode arranged oppositely; a channel placed on the electrode to limit the flow of fluid; the starting end of the channel has multiple inlet branch channels, which converge into a main channel, and the end end has multiple outlet branch channels, and a top cover with multiple fluid inlets and outlets is arranged above the channel; a syringe pump is connected to the inlet and outlet of the top cover in the flow electrotransfection device by a pipeline to control the flow rate of the fluid; a voltage source is connected to the electrode by an electrical connector to set and generate a pulse voltage. The flow electrotransfection system uses the fluid channel and the connected syringe pump to realize the continuous flow of various suspensions in the fluid channel, so that the process of cell electrotransfection can be carried out continuously, and a large number of samples can be quickly processed.

[0010] Chinese patent CN201610806987 discloses a disposable for electrotransfection of cells, comprising: a fluid compartment inside the disposable; a first fluid port for providing a cell suspension to the fluid compartment; and a second fluid port for delivering a fluid including at least one compound to be electrotransfected into the cells to the fluid compartment; a first electrode and a second electrode arranged in the fluid compartment; at least one outlet port for delivering the fluid from the fluid compartment, wherein the first fluid port and the second fluid port are fluidically connected to a mixing channel, and the mixing channel is fluidically connected to the fluid compartment.

[0011] However, the above disclosed flow electrotransfection devices will generate a large number of bubbles during the actual electrotransfection process. The bubbles attached to the electrode plate affect the uniformity of the electric field, resulting in unstable electrotransfection effect. The inventor has made some efforts to overcome the generation of bubbles during electrotransfection by pressurizing the electrotransfection liquid. For example, CN2018104939356 discloses an intermittent flow electrotransfection device; CN2018110783561 discloses an intermittent flow electrotransfection device; CN2018109939121 discloses a flow electrotransfection device; although the above patents have achieved certain technical effects, the above patents do not affect the present invention.

[0012] At the same time, the inventors also found that during the electrofection process, a paste would remain on the tube wall, affecting the electric field strength and uniformity. After searching, the inventors found that in the field of flow electrofection, the technical problems of generating bubbles and removing the generated paste during the electrofection process still need to be solved. Summary of the invention

[0013] The present invention aims at solving the technical problems existing in the above-mentioned prior art. According to the principle of water pressure well, the present invention provides an electroporation device which can automatically inject liquid and pressurize liquid through a movable piston, thereby suppressing the generation of bubbles in the cell fluid during electroporation and effectively removing the paste remaining on the tube body.

[0014] Another object of the present invention is to improve the stability of the electroporation device and to increase the efficiency of electroporation.

[0015] In order to achieve the above-mentioned object, the technical solution of the electroporation device provided by the present invention is summarized as follows:

[0016] An electroporation device comprises a tube body, a first electrode, a second electrode, a piston, and a gasket, wherein the first electrode and the second electrode are placed parallel to the inner wall of the tube body, a sealed end is provided at one end of the tube body, a piston capable of relative movement along the tube body is provided at the other end of the tube body, the piston comprises a plug and a connecting rod, the plug is fixedly connected to the gasket, the gasket and the tube body have a cavity for accommodating a liquid sample, the tube body is provided with at least one liquid inlet and / or outlet and at least one liquid inlet and / or outlet valve, the liquid inlet and / or outlet is located at the sealed end of the tube body, and the liquid inlet and / or outlet valve is located at the liquid inlet and / or outlet of the tube body.

[0017] Preferably, the cross-sectional area of ​​the plug is smaller than the cross-sectional area of ​​the cavity, and more preferably, the cross-sectional diameter or side length of the plug is smaller than the cross-sectional diameter or side length of the cavity; the cross-sectional profile of the plug is the same as the cross-sectional profile of the cavity. The plug is made of a hard solid material, which is selected from insulating materials such as ceramics and plastics.

[0018] Preferably, the cross-sectional area of ​​the gasket is not less than the cross-sectional area of ​​the cavity, and further preferably, the cross-sectional area of ​​the gasket is the same as the cross-sectional area of ​​the cavity, and the contour shape of the gasket cross section is the same as the contour shape of the cavity cross section. It is further preferred that the cross-sectional diameter or side length of the gasket is not less than the cross-sectional diameter or side length of the cavity, and further preferably, the cross-sectional diameter or side length of the gasket is equal to the cross-sectional diameter or side length of the cavity. The gasket material is a flexible soft material, and the flexible soft material is a deformable liquid-impermeable insulating material selected from rubber, silicone, plastic, etc. The thickness of the gasket is not less than the difference between the cross-sectional area of ​​the cavity and the cross-sectional area of ​​the plug, and further preferably, the thickness of the gasket is equal to the difference between the cross-sectional area of ​​the cavity and the cross-sectional area of ​​the plug. It is further preferred that the thickness of the gasket is not less than the difference between the cross-sectional diameter or side length of the cavity and the cross-sectional diameter or side length of the plug, and further preferably, the thickness of the gasket is equal to the difference between the cross-sectional diameter or side length of the cavity and the cross-sectional diameter or side length of the plug.

[0019] Preferably, the plug and the gasket are fixedly connected, and the fixed connection refers to connection through a chemical agent (such as an adhesive), or the plug and the gasket are connected by a physical fixing method.

[0020] Preferably, the lengths of the first electrode and the second electrode are smaller than the length of the tube body, and one end of the first electrode and the second electrode are flush with the sealed end of the tube body.

[0021] Preferably, the piston is set at a working position, which is the position where the first electrode and the second electrode are away from the other end of the sealing end of the tube body, that is, the height of the liquid sample to be processed in the cavity is flush with the upper ends of the first electrode and the second electrode.

[0022] Preferably, when the liquid inlet and / or outlet valve is closed, the gasket and the interior of the tube body form a closed cavity.

[0023] Preferably, the device also includes a push-pull device that enables the piston to move relatively along the tube body, and the push-pull device is connected to the connecting rod; the push-pull device includes but is not limited to a hydraulic rod, a pneumatic rod, a spiral push-pull device, an electromagnetic push-pull device, a human push-pull device, etc.

[0024] Preferably, the device further comprises a conduit, wherein the conduit is sealingly connected to the liquid inlet and / or outlet.

[0025] Beneficial effects brought by the technical solution of the present invention:

[0026] The electroporation device of the present invention can continuously process a large amount of liquid samples and can effectively remove the paste remaining in the tube body, thereby improving the electroporation efficiency;

[0027] The electroporation device of the present invention pressurizes the liquid during the electroporation process, inhibits the generation of bubbles, reduces the non-uniformity of the electric field strength caused by the attachment of bubbles, improves the stability of the electroporation device, and thus improves the electroporation efficiency.

[0028] The electroporation device of the present invention transplants the principle of a water pressure well, has a novel structural design, can automatically feed liquid, and is groundbreaking. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 An example diagram of the overall structure of the electroporation device of the present invention;

[0030] Figure 2 Schematic diagram of liquid entering the cavity of the electroporation device of the present invention;

[0031] Figure 3 Schematic diagram of the electroporation device of the present invention performing pressurized electroporation after the liquid completely enters the cavity;

[0032] Figure 4 A schematic diagram of the electroporation device of the present invention for removing liquid after electroporation is completed;

[0033] Notes on the figures: 1-valve, 2-sealing end of the tube body, 3-gasket, 4-plug, 5-connecting rod, 6-first electrode, 7-second electrode, 8-cavity, 9-tube body. Implementation

[0034] Embodiment 1

[0035] Overall structural design (I)

[0036] The electroporation device provided by the present invention is as follows Figure 1As shown, the device comprises a tube body (9), wherein one end of the tube body (9) is open and the other end is provided with a tube body sealing end (2); the device also comprises a first electrode (6) and a second electrode (7), wherein the length of the first electrode (6) and the second electrode (7) is less than the length of the tube body (9), and one end of the first electrode (6) and the second electrode (7) is flush with the tube body sealing end (2); the tube body sealing end (2) is provided with a valve (1) for controlling the inflow and outflow of liquid; the device also comprises a gasket (3), a plug (4) and a connecting rod (5), wherein the plug (4) and the gasket (3) are connected to each other. ) are fixedly connected by an adhesive, the gasket (3) and the tube body (9) have a cavity (8) for accommodating a liquid sample inside, the cross-sectional diameter or side length of the plug (4) is smaller than the cross-sectional diameter or side length of the cavity (8); the cross-sectional profile of the plug (4) is the same as the cross-sectional profile of the cavity (8), and the material of the plug (4) is ceramic; the cross-sectional diameter or side length of the gasket (3) is the same as the cross-sectional diameter or side length of the cavity (8), and the cross-sectional profile of the gasket (3) is the same as the cross-sectional profile of the cavity (8). The gasket material is rubber. The thickness of the gasket (3) is equal to the difference between the cross-sectional diameter or side length of the cavity (8) and the cross-sectional diameter or side length of the plug (4); the connecting rod (5) is capable of moving the plug (4) and the gasket (3) up and down. When the gasket (3) moves up and down with the connecting rod (5), the gasket (3) and the outer wall of the cavity (8) rub against each other to produce a deformation in the opposite direction of the movement. When the gasket (3) is deformed toward the plug (4) due to the movement, the gasket (3) can fill the gap between the plug (4) and the cavity (8), so that the cavity (8) forms a sealed state.

[0037] Embodiment 2

[0038] How to use the device (Part 2)

[0039] The electroporation device provided by the present invention is used as follows Figure 2-4 As shown, first, the sample is injected. The valve (1) is opened, and the connecting rod (5) is pushed to move the plug (4) and the gasket (3) to the sealed end (2) of the tube body; the liquid sample to be processed is added to the top of the plug (4) and the gasket (3) through a pipette; the valve (1) is closed, and the connecting rod (5) is pulled to move the plug (4) and the gasket (3) to a working position away from the sealed end (2) of the tube body. At this time, the air pressure in the cavity (8) is less than the external air pressure, and the gasket (3) is deformed toward the cavity (8), as shown in FIG. Figure 2 As shown, the liquid sample to be processed added above the plug (4) and the gasket (3) enters the cavity (8) through the gap between the gasket (3) and the tube wall under the influence of air pressure.

[0040] When the height of the liquid sample to be processed in the cavity (8) is flush with the upper ends of the first electrode (6) and the second electrode (7), the piston is in the working position. Figure 3As shown, by pushing the connecting rod (5), the gasket (3) is deformed toward the side of the plug head (4), and the gasket (3) fills the gap between the plug head (4) and the cavity (8) so that the cavity (8) is sealed. At the same time, by pushing the connecting rod (5), the cavity (8) filled with liquid is pressurized.

[0041] Electroporation is performed while applying pressure. After electroporation is completed, Figure 4 , open the valve (1), push the connecting rod (5) to make the plug (4) and the gasket (3) at the sealing end (2) of the tube body, and the processed liquid sample is discharged from the cavity (8). Repeat the above operation to process the sample again.

[0042] Embodiment 3

[0043] Bioassay experiments

[0044] CHO-S cells (Chinese hamster ovary cells) in the logarithmic growth phase were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in electrotransfection buffer to a cell density of 1×10 7 ug / ml, add the plasmid pCDNA3.1-GFP to be electroporated into the cells to make the concentration of the plasmid 20 μg / ml, and mix gently.

[0045] The electrotransfection device prepared in Example 1 of the present invention was used for experiments. The hydraulic rod was used to control the movement of the piston between the working position and the non-working position. The electrotransfection instrument was used to perform electrical stimulation under the following conditions: voltage of 180 volts, pulse width of 6 milliseconds, pulse number of 2 times, and pulse interval of 1 second.

[0046] The comparative experiment was to directly perform electrotransfection after the liquid filled the cavity to the piston working position without applying pressure to the liquid in the cavity.

[0047] The test of the technical solution of the present invention is to perform electrotransfection after the liquid fills the cavity to the working position of the piston and applies pressure to the liquid in the cavity.

[0048] After electroporation, the transfected cell suspension was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, CD-OptiCHO medium was added to resuspend the cells, and the cells were inoculated in a conical flask for culture at a density of 2×10 6 / ml, and then the conical flask was placed on a shaker for culture at a speed of 125 rpm. The culture conditions were: temperature of 37 degrees Celsius and carbon dioxide concentration of 5%. After 24 hours, the cells were observed under a fluorescence microscope, and the electrotransfection efficiency and cell survival rate were detected by flow cytometry.

[0049] Through observation of the electrotransfection process, the comparative experiment showed that a small amount of bubbles were generated near the electrode during the electrotransfection process, affecting the electrotransfection effect; while no bubbles were generated during the electrotransfection process in the experiment of the technical solution of the present invention.

[0050] By comparing the results of electrotransfection, the electrotransfection efficiency of the comparative test was 60%-62%, and the cell survival rate was 81%-86%; the electrotransfection efficiency of the technical solution test of the present invention was 86%-88%, and the cell survival rate was 82%-84%. The experimental results show that the electrotransfection device provided by the technical solution test of the present invention has a higher electrotransfection efficiency.

[0051] Although the present invention has been described in detail, modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. It should be understood that the aspects of the present invention described above and / or in the appended claims and parts of the various embodiments and various features may be combined or interchanged in whole or in part. As will be appreciated by those skilled in the art, in the description of the aforementioned various embodiments, those embodiments referring to another embodiment may be appropriately combined with other embodiments. In addition, it will be appreciated by those of ordinary skill in the art that the aforementioned description is by way of example only and is not intended to limit the present invention.

Claims

1. An electroporation device, comprising a tube, a first electrode, a second electrode, a piston, and a gasket, characterized in that: The first electrode and the second electrode are placed parallel to the inner wall of the tube body, a sealed end is provided at one end of the tube body, a piston capable of relative movement along the tube body is provided at the other end of the tube body, the piston comprises a plug and a connecting rod, the plug is fixedly connected with the gasket, the gasket and the tube body have a cavity for accommodating a liquid sample, the tube body is provided with at least one liquid inlet and / or outlet and at least one liquid inlet and / or outlet valve, the liquid inlet and / or outlet is located at the sealed end of the tube body, and the liquid inlet and / or outlet valve is located at the liquid inlet and / or outlet of the tube body; The cross-sectional area of ​​the plug is smaller than the cross-sectional area of ​​the cavity; The cross-sectional area of ​​the gasket is not less than the cross-sectional area of ​​the cavity; The thickness of the gasket is not less than the difference between the cross-sectional diameter or side length of the cavity and the cross-sectional diameter or side length of the plug.

2. The electroporation device according to claim 1, characterized in that The cross-sectional shape of the gasket is the same as the cross-sectional shape of the cavity.

3. The electroporation device according to claim 1, characterized in that The gasket material is a flexible material.

4. The electroporation device according to claim 3, characterized in that The flexible material is selected from rubber, silicone and plastic.

5. The electroporation device according to claim 1, characterized in that: The plug head is made of hard material.

6. The electroporation device according to claim 5, characterized in that The hard material is selected from ceramics and plastics.

7. The electroporation device according to claim 1, characterized in that: The thickness of the gasket is equal to the difference between the cross-sectional diameter or side length of the cavity and the cross-sectional diameter or side length of the plug.

8. The electroporation device according to claim 1, characterized in that The length of the first electrode and the second electrode is smaller than the length of the tube body.

9. The electroporation device according to claim 1, characterized in that: When the liquid inlet and / or outlet valve is closed, the gasket and the interior of the tube body form a closed cavity.

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