CN110903970A

CN110903970A - Intermittent flow type formula electrotransfection device

Info

Publication number: CN110903970A
Application number: CN201811078356.1A
Authority: CN
Inventors: 戴晓兵
Original assignee: Etta Biotech Co Ltd
Current assignee: Etta Biotech Co Ltd
Priority date: 2018-09-17
Filing date: 2018-09-17
Publication date: 2020-03-24

Abstract

The invention discloses an intermittent flow type electrotransfection device which comprises a tube body, a first electrode, a second electrode and a piston, and is characterized in that the first electrode and the second electrode are arranged on the inner wall of the tube body in parallel, a sealing end is arranged at one end of the tube body, the piston capable of moving along the tube body relatively is arranged at the other end of the tube body, a cavity for containing a liquid sample is arranged inside the piston and the tube body, the tube body is provided with at least one liquid inlet and outlet and at least one liquid inlet and outlet two-way valve, the liquid inlet and outlet is positioned at the sealing end of the tube body, and the liquid inlet and outlet two-way valve is positioned at the liquid inlet and outlet of the tube body. The invention discloses a flow type electrotransfection device, which provides a device for electrotransfection of a sample solution with large volume, and inhibits bubbles generated in the electrotransfection process by a movable piston pressurization mode, and improves the uniformity of an electric field, thereby improving the stability of the electrotransfection device and the electrotransfection efficiency.

Description

Intermittent flow type formula electrotransfection device

Technical Field

The invention relates to the field of electrotransfection, in particular to an intermittent flow type electrotransfection device, which inhibits bubbles generated by electrotransfection in a piston moving pressurization mode.

Background

The cell membrane is a thin membrane surrounding the cell periphery and is a permeable barrier for selective exchange of substances between the cell and the outside. The cell membrane makes the cell an independent life unit and has a relatively stable internal environment. Some substances in the surrounding environment may pass through the cell membrane, others do not. Cells can take up nutrients from the surrounding environment through the cell membrane, excrete metabolites, and allow the transport of substances to reach an equilibrium state. Therefore, the basic function of the cell membrane is to maintain a relatively stable intracellular microenvironment and selectively exchange substances with the external environment.

It is found that if a certain intensity of electric stimulation is applied to cells for a certain period of time, micropores can be induced on cell membranes, so that the permeability of the cells is enhanced, and the cell Electroporation (Electroporation) refers to a biophysical process of the cells under the action of an applied pulse electric field, wherein transient micropores are formed on cell membrane lipid bilayers. Electrotransfection (Electrotransfection) is a technique for introducing foreign biological macromolecules, such as DNA, RNA or proteins, into cells using electroporation. When the cell membrane is subjected to electroporation, the permeability and the membrane conductance of the cell membrane are increased instantaneously, so that molecules, such as hydrophilic molecules, DNA, proteins, virus particles, drug particles and the like, which cannot pass through the cell membrane under normal conditions can enter the cell through micropores. After the electrical stimulation is removed in a short time, the micropores in the cell membrane disappear, and the cell membrane becomes a selective permeability barrier again.

Compared with the traditional chemical transfection and virus transfection, the electrotransfection has the advantages of no chemical pollution, no permanent damage to cells, high efficiency and the like, and has wide application prospect in the fields of biophysics, molecular biology, clinical medicine and the like.

Although the mechanism of electrotransfection is not completely understood, it is well known in this context that cell electrotransfection involves the movement of the lipid bilayer of the cell membrane, resulting in the formation of transient micropores in the membrane, allowing exogenous molecules to enter the cell through the micropores.

In the prior art, there are three main types of methods for completing the process of electrotransfection of cells:

the cells are placed between a pair of parallel electrodes spaced a few millimeters to a few centimeters apart. The cells are electrically stimulated in an electric field between the electrodes for the purpose of electrotransfection. For example, US patent No. 5389069.

The micro needle electrode is pricked into tissue or cell fluid to shock the cell electrically to reach the aim of electrotransfection. For example, US patent No. 5389069.

A chamber is placed between a pair of parallel electrodes so that a suspension solution of cells is electrically shocked while flowing in the chamber. For example, US patent US 6773669.

Chinese patent CN201010242144 discloses a flow electrotransfection device and system, the system includes: a flow electrotransfection device comprising: the electrode is arranged in parallel and in pairs, and each pair of electrodes comprises an anode and a cathode which are oppositely arranged; a channel disposed over the electrode that restricts fluid flow; the starting end of the channel is provided with a plurality of inlet branch channels which are converged into a main channel, the ending end of the channel is provided with a plurality of outlet branch channels, and a top cover with a plurality of fluid inlets and outlets is arranged above the channel; the injection pump is connected to the inlet and the outlet of the top cover in the flow type electrotransfection device through pipelines to control the flow rate of the fluid; and the voltage source is connected with the electrode set by the electric connector and generates pulse voltage. The flow-type electrotransfection system utilizes the fluid channel and the connected injection pump to realize the continuous flow of various suspensions in the fluid channel, thereby enabling the process that the cells are electrotransfected to be continuously carried out and realizing the rapid processing of a large number of samples.

Chinese patent CN201610806987 discloses a disposable article for the 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 comprising at least one compound to be electrotransfected into the cell to the fluid compartment; a first electrode and a second electrode disposed in the fluid compartment; at least one outlet port delivering the fluid from the fluid compartment, wherein the first and second fluid ports are in fluid communication with a mixing channel in fluid communication with the fluid compartment.

However, the flow electrotransfection device disclosed above generates a large amount of bubbles during the actual electrotransfection process, and the generated bubbles adhere to the electrode plates to affect the uniformity of the electric field, thereby causing the electrotransfection effect to be unstable. The inventor finds that the technical problem of bubble generation in the electrotransfection process is not solved in the field of flow electrotransfection.

Disclosure of Invention

The invention provides an intermittent flow type electrotransfection device which can restrain bubbles generated in the electrotransfection process in a mode of pressurizing by a movable piston, aiming at the technical problems in the prior art. It is another object of the present invention to improve the stability of electrotransfection devices and to improve the efficiency of electrotransfection.

In order to achieve the above object, the technical scheme of the intermittent flow type electrotransfection device provided by the invention is summarized as follows: an intermittent flow type electrotransfection device comprises a tube body, a first electrode, a second electrode and a piston, and is characterized in that the first electrode and the second electrode are arranged on the inner wall of the tube body in parallel, a sealing end is arranged at one end of the tube body, the piston capable of moving relative to the tube body is arranged at the other end of the tube body, a cavity for containing a liquid sample is arranged inside the piston and the tube body, the tube body is provided with at least one liquid inlet and outlet and at least one liquid inlet and outlet two-way valve, the liquid inlet and outlet is located at the sealing end of the tube body, and the liquid inlet and outlet two-way valve is located at the liquid inlet and outlet of the tube body. The liquid inlet and outlet two-way valve controls the opening and closing of the liquid inlet and outlet.

Preferably, the tube body is provided with at least two liquid inlets and outlets, the liquid inlets and outlets can be liquid inlets or liquid outlets, and at least two liquid inlet and outlet two-way valves are correspondingly arranged; the liquid inlet and outlet two-way valve is used for controlling liquid sample introduction and liquid sample discharge; further preferably, the tube body is provided with at least one liquid inlet and one liquid outlet, and at least two liquid inlet and outlet two-way valves; it is further preferred that the fluid inlet and outlet is located at the sealed end of the body opposite the piston.

Preferably, the cross section of the piston is the same as or close to that of the inside of the tube body, and further preferably, the cross section of the inside of the tube body is square, and can also be round or in other shapes;

preferably, a sealing device is arranged at the contact position of the piston and the pipe body, and the sealing device is preferably a sealing gasket, a sealing rubber strip or other devices with sealing performance and the like;

preferably, when the liquid inlet and outlet two-way valve and the vent valve are closed, the piston and the interior of the tube body form a closed cavity;

preferably, when the liquid inlet and outlet two-way valve is closed, the pressure value inside the closed cavity is 1.0-2.5Mpa after the piston moves relative to the pipe body; further preferably, the pressure value in the cavity is 1.1-2.0 Mpa; further preferably, the pressure value in the cavity is 1.2-1.5 Mpa;

preferably, the device also comprises a plurality of guide pipes which are divided into a liquid inlet pipe and a liquid outlet pipe, and the guide pipes and the pipe body are arranged in a sealing way;

preferably, the device further comprises a push-pull device enabling the piston to move relatively along the tube, and the push-pull device is of a type including but not limited to a hydraulic rod, a pneumatic rod, a spiral push-pull device, an electromagnetic push-pull device, and the like;

preferably, the piston is provided with a working position and a non-working position, the piston moves from the non-working position to the working position and can suck liquid into the cavity from the liquid inlet and the liquid outlet, and the piston moves from the working position to the non-working position and can provide pressure for the interior of the cavity;

the preparation method of the device comprises the following steps: the method comprises the steps of firstly preparing a tube body, selecting plastic capable of being cured after heating as a material of the tube body, preparing the tube body by utilizing a die for preparing the tube body, respectively arranging a liquid injection port and a liquid outlet port on the tube body, and parallelly arranging two electrodes with the same shape and size on two opposite sides of the inner wall of the tube body. One end of the tube body is set to be a sealing end, a piston parallel to the sealing end face of the tube body is arranged at the opening of the other end of the tube body, a sealing rubber strip is adopted to seal the contact position of the piston and the tube body, and one side, which is not in contact with the cavity, of the piston is connected with the push-pull device. The sealed end of the tube body is provided with a liquid inlet and a liquid outlet. The liquid inlet and the liquid outlet are provided with guide pipes which are connected with the pipe body in a sealing way, and the guide pipes are provided with two-way valves.

The technical scheme of the invention has the following beneficial effects:

the intermittent flow electrotransfection device of the present invention is capable of processing large volumes of liquid samples.

The intermittent flow type electrotransfection device pressurizes liquid in the electrotransfection process, inhibits bubbles from generating, reduces the nonuniformity of electric field intensity caused by bubble attachment, and improves the stability of the electrotransfection device, thereby improving the electrotransfection efficiency.

The intermittent flow type electrotransfection device has novel structural design and is pioneering.

Drawings

FIG. 1 is an exemplary diagram of an intermittent flow electrotransfection apparatus of the present invention;

the attached drawings are annotated: 1-a first electrode, 2-a second electrode, 3-a tube body, 4-a sealing end, 5-a liquid inlet tube, 6-a liquid outlet tube, 7-a first two-way valve, 8-a second two-way valve, 9-a push-pull device, 10-a piston non-working position, 11-a piston first working position and 12-a piston second working position.

Detailed description of the preferred embodiments

Example one

Overall structure design 1

As shown in figure 1, the intermittent flow type electrotransfection device provided by the invention is characterized in that a first electrode (1) and a second electrode (2) are respectively arranged on the inner wall of a tube body (3) in parallel, a tube orifice at one end of the tube body (3) is set to be a sealing end (4), a liquid inlet tube (5) and a liquid outlet tube (6) are arranged on the sealing end (4) of the tube body, a first bidirectional valve (7) is arranged in the liquid inlet tube (5) and close to the interface between the liquid inlet tube (5) and the sealing end (4), and a second bidirectional valve (8) is arranged in the liquid outlet tube (6) and close to the interface between the liquid outlet tube (6) and the sealing end (4). The piston is arranged at the opening of the other end of the tube body (3), the piston is fixedly connected with the push-pull device (9), the push-pull device (9) can move the piston from a piston non-working position (10) to a piston first working position (11) or a piston second working position (12), and the push-pull device (9) is preferably provided with a hydraulic rod to push the piston to enable liquid carried in the cavity to bear certain pressure. And applying electric pulses to the first electrode (1) and the second electrode (2), wherein the electric pulses enable an electric field to be formed in the cavity, so that the cell membranes form micropores, and the target substances in the liquid sample can enter the cells. After the electrotransfection is finished, the push-pull device (9) can move the piston from the second working position (12) of the piston to the non-working position (10) of the piston, and liquid in the tube body (3) is extruded out of the tube body. The above procedure was repeated to perform intermittent flow electrotransfection.

Example two

Intermittent liquid feeding mode and pressurizing mode

When liquid is fed, the first two-way valve (7) is opened, the second two-way valve (8) is closed, the piston is pulled to the first working position (11) of the piston from the non-working position (10) of the piston by the push-pull device (9), and a target liquid sample enters the cavity from the liquid inlet pipe (5); closing the first two-way valve (7), pushing the piston from a first working position (11) to a second working position (12) by a push-pull device (9), and pressurizing liquid in the cavity; the electrotransfection of the liquid is carried out in a pressurized state. After the electrotransfection is finished, a second bidirectional valve (8) is opened, the push-pull device (9) pushes the piston from the second working position (12) of the piston to the non-working position (10) of the piston, and the liquid in the tube body is pushed out of the tube body. Repeating the above liquid inlet and outlet actions to realize the electrotransfection of a large amount of liquid.

EXAMPLE III

Bioassay experiments

CHO-S cells (Chinese hamster ovary cells) in logarithmic growth phase were collected, centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells resuspended in electrotransfection buffer to a cell density of 1X10⁷One/ml, adding plasmid pCDNA3.1-GFP to be transfected into cells to make the concentration of the plasmid be 20 microgram/ml, and mixing gently.

The electrotransfection device prepared by the first embodiment of the invention is used for experiments, the hydraulic rod is used for controlling the piston to move between the working position and the non-working position, the electrotransfection device is matched for use, and the following conditions are adopted for carrying out electrostimulation: voltage 180 volts, pulse width 6 milliseconds, number of pulses 2, pulse interval 1 second.

In the contrast test, pressure is not applied to liquid in the cavity, the piston is kept at a first working position (11) of the piston, and after the cavity is filled with the liquid, electrotransfection is directly carried out;

the experiment of the technical scheme of the invention is to apply pressure to liquid in the cavity, and after the cavity is filled with the liquid, the piston is moved from the first working position (11) of the piston to the second working position (12) of the piston through the hydraulic rod, so that the liquid is pressurized and electrotransfection is carried out.

After the end of the electrotransfection, the transfected cell suspension was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. Discarding supernatant, adding CD-OptiCHO culture medium to suspend cells, inoculating into a triangular conical flask, and culturing at a culture density of 2 × 10⁶And (2) culturing the cells in a triangular conical flask on a shaking table at the rotating speed of 125 revolutions per minute under the culture conditions: the temperature was 37 ℃ and the carbon dioxide concentration was 5%. After 24 hours, the cells were observed under a fluorescence microscope, and the electrotransfection efficiency and the cell viability were measured by a flow cytometer.

Through observation of the electrotransfection process, a contrast test can generate a small amount of bubbles near the electrode in the electrotransfection process to influence the electrotransfection effect; the experiment of the technical scheme of the invention does not generate bubbles in the electrotransfection process.

By comparing the electrotransfection results, the electrotransfection efficiency of a comparative test is 60-64%, and the cell survival rate is 70-83%; the electrotransfection efficiency tested by the technical scheme of the invention is 84-86%, and the cell survival rate is 82-85%. Experimental results show that the electrotransfection device provided by the technical scheme of the invention has higher electrotransfection efficiency and cell survival rate.

Although the present invention has been described in detail, modifications within the spirit and scope of the invention will be apparent to those skilled in the art. It should be understood that aspects of the invention and portions of the various embodiments and various features described above and/or in the appended claims may be combined or interchanged either in whole or in part. As will be appreciated by one skilled in the art, in the foregoing description of the various embodiments, those embodiments referring to another embodiment may be combined with other embodiments as appropriate. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.

Claims

1. An intermittent flow type electrotransfection device comprises a tube body, a first electrode, a second electrode and a piston, and is characterized in that the first electrode and the second electrode are arranged on the inner wall of the tube body in parallel, a sealing end is arranged at one end of the tube body, the piston capable of moving relative to the tube body is arranged at the other end of the tube body, a cavity for containing a liquid sample is arranged inside the piston and the tube body, the tube body is provided with at least one liquid inlet and outlet and at least one liquid inlet and outlet two-way valve, the liquid inlet and outlet is located at the sealing end of the tube body, and the liquid inlet and outlet two-way valve is located at the liquid inlet and outlet of the tube body.

2. An intermittent flow electrotransfection device as claimed in claim 1, wherein the tube is provided with at least two fluid inlet and outlet ports and at least two fluid inlet and outlet two-way valves.

3. An intermittent flow electrotransfection device according to claim 1, wherein the piston is disposed in an operative position and an inoperative position.

4. An intermittent flow electrotransfection device according to claim 1, wherein a sealing means is provided where the piston contacts the tube, preferably a sealing gasket, sealing strip or other means with sealing properties.

5. An intermittent flow electrotransfection device according to claim 1, wherein the piston and the interior of the tube form a sealed chamber when the liquid inlet and outlet two-way valve is closed.

6. An intermittent flow electrotransfection device according to claim 5, wherein the pressure inside the closed chamber is 1.0-2.50MPa, preferably 1.1-2.0MPa, after the piston moves relatively along the tube when the liquid inlet and outlet two-way valve is closed.

7. An intermittent flow electrotransfection device according to claim 1, further comprising a push-pull device to enable relative movement of the plunger along the tube.

8. An intermittent flow electrotransfection device according to claim 7, wherein the push-pull device includes, but is not limited to, hydraulic rod, pneumatic rod, screw push-pull device, electromagnetic push-pull device, and the like.

9. An intermittent flow electrotransfection device according to claim 1, further comprising a conduit in sealed connection with the liquid inlet and outlet.