CN111808885A

CN111808885A - Electrotransfection device utilizing air bag pressurization

Info

Publication number: CN111808885A
Application number: CN201910275528.2A
Authority: CN
Inventors: 戴晓兵; 朱士英
Original assignee: Etta Biotech Co Ltd
Current assignee: Etta Biotech Co Ltd
Priority date: 2019-04-12
Filing date: 2019-04-12
Publication date: 2020-10-23

Abstract

The invention provides an electrode cup for pressurizing through airbag expansion, which comprises a cup body, a cup cover, a planar electrode, an airbag pressurizing device and an air pump, wherein the airbag pressurizing device is positioned at the bottom of the cup body or inside the cup cover; the electrode cup provided by the invention can inhibit the generation of bubbles in the electrotransfection process and improve the electrotransfection efficiency.

Description

Electrotransfection device utilizing air bag pressurization

Technical Field

The invention relates to the field of electrotransfection, in particular to a pressurized electrotransfection device, and more particularly relates to an electrotransfection device for inhibiting bubble generation in liquid in an electrotransfection process in a pressurizing mode through air bag expansion.

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 the cell suspension is 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 cell 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 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 instability of the electrotransfection effect. The present inventors have made some efforts to reduce the generation of bubbles during electrotransfection by pressurizing the suspension of the electrotransfected cells, for example chinese patent CN 20181049356 discloses an intermittent flow electrotransfection device; CN2018110783561 discloses an intermittent flow type electrotransfection device; CN2018109939121 discloses a flow-type electrotransfection device; although the above patent has achieved certain technical effects, the above patent does not affect the present invention.

Disclosure of Invention

The invention provides an electrotransfection device for inhibiting bubble generation in the electrotransfection process in a mode of inflating and pressurizing an air bag aiming at the technical problems in the prior art.

Another object of the present invention is to improve the stability of electrotransfection devices and to improve the electrotransfection efficiency.

Drawings

FIG. 1 is a view showing an example of the structure of an airbag pressurizing apparatus of the present invention in the bottom of a cup body;

FIG. 2 is a schematic view of the air bag pressurizing apparatus of the present invention performing pressurized electrotransfection after liquid has completely entered the cup body;

FIG. 3 is a view showing an example of the structure of the pressurizing device for an air bag of the present invention in the bottom of a cup body;

FIG. 4 is a schematic view of the airbag pressurizing device of the present invention performing pressurized electrotransfection after liquid has completely entered the cup;

FIG. 5 is a schematic view showing an exemplary structure of the air bag pressurizing device inside the cup cover;

FIG. 6 is a schematic view of the airbag pressurizing device of the present invention performing pressurized electrotransfection after liquid has completely entered the cup;

the attached drawings are annotated: the device comprises a cup cover 1, a cup body 2, a first electrode 3, a limiting plate 4, a conduit 5, a cell suspension 6, a second electrode 7, an air bag 8 and an air pump 9.

Detailed Description

The invention provides an electrode cup which comprises a cup cover (1), a cup body (2), a first electrode (3), a second electrode (7), an air bag (8) and an air pump (9), wherein the first electrode (3) and the second electrode (7) are positioned on the side wall of the cup body (2), and as shown in figures 1-4, the air bag (8) is positioned at the bottom of the cup body (2); or as shown in fig. 5-6, the air bag (8) is positioned inside the cup cover (1); the air bag (8) is connected with the air pump (9) through a conduit (5), and the conduit (5) is hermetically connected with the bottom of the cup body (2) or the top of the cup cover (1).

The cup body (2) is connected with the cup cover (1) through a buckle, so that a sealed cavity is formed inside the electrode cup. The cup cover (1) and the cup body (2) can be connected through an embedded structure (shown in figures 1-2 and 5-6) or an outer sleeve structure (shown in figures 3-4), and the embedded structure or the outer sleeve structure refers to the position relation of the cup cover (1) relative to the cup body (2) when the cup body (2) and the cup cover (1) are connected; the embedded structure is that the outer diameter of the bottom part of the cup cover (1) is consistent with the inner diameter of the cup body (2), the outer side of the cup cover (1) and the cup body (2) are respectively provided with a bulge or groove structure, when the bottom part of the cup cover (1) is inserted into the cup body, the bulge structure on the cup cover (1) is matched with the groove on the cup body (2) to form a closed cavity; the outer sleeve type structure is that the inner diameter of the bottom part of the cup cover (1) is consistent with the outer diameter of the cup body (2), the outer side of the cup cover (1) and the cup body (2) are respectively provided with a bulge or groove structure, and when the bottom part of the cup cover (1) is buckled on the cup body, the bulge structure on the cup body (2) is inosculated with the groove on the cup cover (1) to form a closed cavity.

The cup body (2) and the cup cover (1) can be made of insulating materials, and can be made of plastics, rubber or ceramics.

The electrode cup provided by the invention also comprises a limiting plate (4), wherein the size of the limiting plate (4) is the same as that of the cross section of the inner part of the cup body (2). The limiting plate (4) has the main functions of: the contact surface of the air bag (8) and the cell suspension (6) can keep a plane in the expanded state, thereby ensuring the uniformity of the electric field.

Under this effect prerequisite, the limiting plate has two kinds of condition: firstly, the limiting plate (4) is connected with the air bag (8) and can move along with the contraction and expansion of the air bag (8), and the limiting plate is similar to a piston; the limiting plate (4) can be airtight or air-permeable. Secondly, the limiting plate (4) is fixed at a certain position of the cup body (2), and the limiting plate (4) is breathable.

When the air bag (8) is made of certain material with better shaping, the contact surface of the air bag (8) and the cell suspension (6) can automatically keep a plane in an expanded state, and in this case, the limiting plate (4) is not needed.

The air bag (8) is contracted in the initial state and only occupies a small amount of the inner space of the cup body (2).

The initial state of the limiting plate (4) needs to be determined according to the three conditions, and the three conditions are as follows: firstly, when the limiting plate (4) is parallel to the bottom surface of the cup body (2), the initial state is at the bottom of the cup body (2), and the working state is vertically moving from bottom to top; secondly, when the limiting plate (4) is parallel to the bottom surface of the cup body (2), the limiting plate is directly fixed on the cup body (2) and is arranged at the position where the air bag is contacted with the cell suspension (6) when the air bag expands; thirdly, the existence of a limit plate is not required.

When the air bag (8) is in a working state, the air bag (8) is expanded to fill the bottom of the cup body (2); when the limiting plate (4) is connected with the air bag (8), the limiting plate (4) is pushed to move the cell suspension (6) upwards integrally; when the limiting plate (4) is fixed on the cup body (2) or the limiting plate is not needed, the air bag (8) directly pushes the cell suspension (6) to integrally move upwards.

When the limiting plate (4) is in a working state, one condition is that the limiting plate is connected with the air bag (8) to play a role similar to a piston; the other condition is that the cell suspension liquid is fixed on the cup body (2), the limiting plate (4) in the working state is not changed, and only along with the expansion of the air bag (8), the cell suspension liquid (6) on one side of the limiting plate (4) adjacent to the air bag (8) is pushed to the upper side of the limiting plate (4).

Limiting plate (4) can be followed cup (2) inner wall and removed, perhaps limiting plate (4) can be fixed at the inside certain position of cup (2).

The limiting plate (4) is fixedly connected with the air bag (8) in a physical mode.

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. The electrode cup comprises a cup body, a cup cover, a planar electrode and an air bag pressurizing device, wherein the planar electrode is positioned on the side wall of the cup body.

2. The electrode cup of claim 1, wherein the balloon pressurization device comprises a balloon and a catheter.

3. The electrode cup of claim 2, further comprising an air pump, wherein the balloon pressurization device is connected to the air pump via a conduit.

4. The electrode cup of claim 3, wherein the conduit is disposed on the bottom of the cup or on the top of the lid, the conduit being in sealing engagement with the bottom of the cup or the top of the lid.

5. The electrode cup according to any one of claims 1 to 4, further comprising a limiting plate having the same size as the cross-section of the inside of the cup body.

6. The electrode cup according to claim 5, wherein the limiting plate is a gas permeable plate or a gas impermeable plate.

7. The electrode cup as claimed in claim 6, wherein the limiting plate is movable along the inner wall of the electrode cup.

8. The electrode cup as claimed in claim 5, wherein the stopper plate is fixed at a position inside the cup body.

9. The electrode cup according to any one of claims 6 to 8, wherein the limiting plate is fixedly connected with the balloon pressurizing device.

10. The electrode cup as claimed in any one of claims 1 to 9, wherein the lid is snap-fitted to the cup body.