A cell electroporator and a method of cell electroporation

Abstract

The application provides a cell electrotransformation instrument and a cell electrotransformation method, which can be applied to the field of bioelectronics and the like, and is used for solving the problem of low reliability of cell electrotransformation. The cell electrotransformation instrument comprises: a first electrode layer and a second electrode layer attached to different surfaces of an insulating layer respectively, the insulating layer has at least one hole, the hole and the surface to which the first electrode layer is attached and the surface to which the second electrode layer is attached form a cavity; a first friction layer is adjacent to the first electrode layer, and a second friction layer is adjacent to the second electrode layer; a rotating device comprises a fixed component and a rotating component, the first electrode layer, the second electrode layer and the insulating layer are fixed to the fixed component, the first friction layer and the second friction layer are fixed to the rotating component, the rotating component drives the first friction layer to rotate at a position with a first gap from the surface of the first electrode layer, and the rotating component drives the second friction layer to rotate at a position with a second gap from the surface of the second electrode layer.

Description

Technical Field

[0001] This application relates to the field of bioelectronics technology, and in particular to a cell electroconversion device and a method for cell electroconversion. Background Technology

[0002] With the continuous development of science and technology, cell electroporation technology has received increasing attention. Cell electroporation technology introduces exogenous macromolecules, such as DNA, RNA, siRNA, and proteins, as well as some small molecules, into the cell membrane. It has significant scientific value in the study of transient or stable protein expression, gene knockout and knock-in, siRNA, and other molecular mechanisms, and is currently used in fields such as cell and molecular biology, immunology, hematology, neurology, cancer research, and new drug development.

[0003] In related technologies, a cell electroporator consists of a pulse generator, an impact chamber, and an electroporation cup. When performing cell electroporation using this cell electroporator, the cell sample needs to be placed in the electroporation cup for reaction. Then, an instantaneous high voltage, such as 3000V, is generated by passing an electric current through the metal electrodes on both sides of the electroporation cup, thereby obtaining the cell electroporation result.

[0004] However, due to the wide variety of cell sizes, the cell electroporation device in related technologies directly applies instantaneous high voltage to the cell sample, which can easily cause some larger cells to die due to the large pores formed on the surface, resulting in a low cell survival rate.

[0005] It is evident that, among related technologies, cell electroporation instruments have relatively low reliability in performing cell electroporation. Summary of the Invention

[0006] This application provides a cell electroporator and a method for cell electroporation to address the problem of low reliability in cell electroporation.

[0007] In a first aspect, a cell electroporation device is provided, comprising a first electrode layer, a second electrode layer, a first friction layer, a second friction layer, an insulating layer, and a rotating device, wherein:

[0008] The first electrode layer and the second electrode layer are respectively attached to different surfaces of the insulating layer, and the insulating layer has at least one hole, which forms a cavity with the surface to which the first electrode layer is attached and the surface to which the second electrode layer is attached;

[0009] The first friction layer is adjacent to the first electrode layer, and the second friction layer is adjacent to the second electrode layer;

[0010] The rotating device includes a fixing component and a rotating component. The first electrode layer, the second electrode layer, and the insulating layer are fixed to the fixing component. The first friction layer and the second friction layer are fixed to the rotating component. The rotating component drives the first friction layer to rotate at a position a first gap away from the surface of the first electrode layer, so that the first electrode layer is positively charged. The rotating component drives the second friction layer to rotate at a position a second gap away from the surface of the second electrode layer, so that the second electrode layer is negatively charged.

[0011] Optionally, the first electrode layer, the second electrode layer, the first friction layer, the second friction layer, and the insulating layer are all annular cylinders with a central hole, and the rotating device is connected to the first electrode layer, the second electrode layer, the first friction layer, the second friction layer, and the insulating layer through each central hole.

[0012] Optionally, the fixing component is a bearing, the rotating component is a bearing pin, the bearing pin is connected to the inner wall of the bearing by a rolling filler, the first electrode layer, the second electrode layer and the insulating layer are fixed to the outer wall of the bearing, and the first friction layer and the second friction layer are fixed to the bearing pin.

[0013] Optionally, the rotating device further includes a pull rope, the bearing pin is a cylinder, and a double hole is provided on the bearing pin with the central axis of the cylinder as the axis of symmetry. The pull rope passes through the double hole, and the pull rope in the torsional state drives the bearing pin to rotate by the action of external forces from opposite directions.

[0014] Optionally, the first electrode layer and the second electrode layer are annular cylinders with a thickness of 1 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm.

[0015] Copper foil is attached to the surface of the insulating layer and the surface adjacent to the first or second friction layer on the first and second electrode layers.

[0016] Optionally, the first friction layer and the second friction layer are annular cylinders with a thickness of 1 mm, an inner diameter of 24 mm, and an outer diameter of 60 mm.

[0017] A nylon film is adhered to the surface of the first friction layer adjacent to the first electrode layer, and a fluorinated ethylene propylene copolymer film is adhered to the surface of the second friction layer adjacent to the second electrode layer.

[0018] Optionally, rabbit fur is adhered to the surface of the first friction layer and the second friction layer adjacent to the first electrode layer or the second electrode layer.

[0019] Optionally, the insulating layer is a cylindrical ring with a thickness in the range of 0.5 to 2 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm, and the inner diameter of the hole is 6 mm.

[0020] Optionally, the rotating device is made of zirconia all-ceramic material.

[0021] In a second aspect, a method for cell electroporation is provided, employing a cell electroporator as described in the first aspect, comprising:

[0022] Cell samples are obtained through at least one chamber;

[0023] The rotating device is controlled to rotate at a gradually increasing speed, thereby causing the first friction layer and the second friction layer to rotate, so that the first electrode layer and the second electrode layer form an electric field in the at least one cavity.

[0024] When it is determined that the charged substance in the solution contained in the cell sample has entered the cell membrane of the cells contained in the cell sample, the rotating device is controlled to stop rotating to obtain the cell electroconversion result.

[0025] In this embodiment, the rotating component drives the first friction layer to rotate at a position a first gap away from the surface of the first electrode layer, and the rotating component drives the second friction layer to rotate at a position a second gap away from the surface of the second electrode layer. This allows the triboelectric effect to be utilized, making the first electrode layer positively charged and the second electrode layer negatively charged. With the first and second electrode layers charged, an electric field is generated in the cavity formed by the holes in the insulating layer and the surfaces to which the first and second electrode layers are attached. After placing a cell sample in the cavity, the cell sample can be electrochemically converted.

[0026] Furthermore, since the first friction layer rotates at a position a first gap away from the surface of the first electrode layer, and the second friction layer rotates at a position a second gap away from the surface of the second electrode layer, the electric field generated by the first electrode layer and the second electrode layer is stabilized, avoiding irreversible damage to the cell membrane during electroconversion treatment, thus preventing low cell survival rate and improving the reliability of cell electroconversion.

[0027] Furthermore, since the layers of the cell electroporator are not glued together, the cell samples inside the chamber can be cleaned by disassembling the cell electroporator, making the operation of the cell electroporator simpler and thus improving the efficiency of cell electroporation to some extent. Attached Figure Description

[0028] Figure 1 A schematic diagram of the principle of a cell electroporator provided in an embodiment of this application;

[0029] Figure 2 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 1 ;

[0030] Figure 3 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 2 ;

[0031] Figure 4 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 3 ;

[0032] Figure 5 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 4 ;

[0033] Figure 6 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 5 ;

[0034] Figure 7 A schematic diagram of the structure of a cell electroporator provided in this application embodiment. Figure 6 . Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0036] The application fields of the cell electroporator provided in the embodiments of this application will be briefly introduced below.

[0037] With the continuous development of science and technology, cell electroporation, also known as cell electroporation, has received increasing attention. Cell electroporation is a technique that introduces exogenous macromolecules, such as DNA, RNA, siRNA, and proteins, as well as some small molecules, into the cell membrane. It has significant scientific value in the study of transient or stable protein expression, gene knockout and knock-in, siRNA, and other molecular mechanisms, and is currently used in fields such as cell and molecular biology, immunology, hematology, neurology, cancer research, and new drug development.

[0038] The basic principle of cell electroporation is that the cell membrane is reversibly perforated by a strong, instantaneous electric field, allowing charged foreign substances in the solution to enter the cell membrane in a manner similar to electrophoresis. Please refer to [reference needed]. Figure 1 (1) is a cell with a cell membrane; under the action of electric field E, the cell membrane begins to form pores. Please refer to [reference needed]. Figure 1(2); When the electric field E is strong enough, reversible small pores appear on the cell membrane. Please refer to [reference needed]. Figure 1 (3) Thus, charged exogenous substances in the solution can diffuse to the cell nucleus and other places. When the voltage formed by the electric field E across the cell is generally 1 to 2V, it can ensure that reversible small holes are made, and that the cell will not die due to membrane damage.

[0039] In related technologies, cell electroporators consist of a pulse generator, an impact chamber, and an electroporation cup. Because the phospholipid bilayer of the cell membrane has a high resistance, the electric field applied across the cell is usually borne by the cell membrane. Therefore, in practice, a parallel electric field is provided to the cell population to generate a voltage of 1–2 V across the cell. Since cells are typically on the micrometer scale, the required electric field usually needs to reach thousands of volts. For example, prokaryotic cells often have a smaller cell radius (approximately 1 μm), requiring a voltage of about 6.6 kV / cm; while eukaryotic cells have a larger cell radius (approximately 25 μm), requiring a voltage of about 260 V / cm, etc.

[0040] When performing cell electroporation using a cell electroporator in related technologies, the cell sample needs to be placed in an electroporation cup for reaction. Then, an instantaneous high voltage, such as 3000V, is generated by passing an electric current through the metal electrodes on both sides of the electroporation cup, thereby obtaining the cell electroporation result.

[0041] While cell electroporation, compared to chemical transfection and viral vector transfection methods, can be used on all eukaryotic cells, including all cell lines, primary cells, stem cells, blood and immune cells, nerve cells, plant cells, and embryos, and offers easier quantitative control, the wide variety of cell sizes means that directly applying a high voltage to the cell sample can easily cause some larger cells to die due to excessively large pores formed on their surface, resulting in low cell viability.

[0042] It is evident that, among related technologies, cell electroporation instruments have relatively low reliability in performing cell electroporation.

[0043] Meanwhile, the cell electroporation instrument in the relevant technology has a relatively complex structure and requires a large number of accessories, which makes the instrument expensive, ranging from tens of thousands to hundreds of thousands of yuan.

[0044] To address the issue of low reliability in cell electroporation using cell electroporators, this application proposes a cell electroporator. Please refer to... Figure 2 The cell electroporator 200 includes a first electrode layer 201, a second electrode layer 202, a first friction layer 203, a second friction layer 204, an insulating layer 205, and a rotating device 206, wherein:

[0045] The first electrode layer 201 and the second electrode layer 202 are respectively attached to different surfaces of the insulating layer 205. The insulating layer 205 has at least one hole, and the hole forms a cavity with the surface to which the first electrode layer 201 and the second electrode layer 202 are attached. The first friction layer 203 is adjacent to the first electrode layer 201, and the second friction layer 204 is adjacent to the second electrode layer 202.

[0046] Please refer to Figure 3 The rotating device 206 includes a fixing component 2061 and a rotating component 2062. A first electrode layer 201, a second electrode layer 202, and an insulating layer 205 are fixed to the fixing component 2061. A first friction layer 203 and a second friction layer 204 are fixed to the rotating component 2062. The rotating component 2062 drives the first friction layer 203 to rotate at a position a first gap away from the surface of the first electrode layer 201, so that the first electrode layer 201 becomes positively charged. The rotating component 2062 also drives the second friction layer 204 to rotate at a position a second gap away from the surface of the second electrode layer 202, so that the second electrode layer becomes negatively charged. The first gap and the second gap can be the same or different.

[0047] In this embodiment, the rotating component drives the first friction layer to rotate at a position a first gap away from the surface of the first electrode layer, and the rotating component drives the second friction layer to rotate at a position a second gap away from the surface of the second electrode layer. This allows the triboelectric effect to be utilized, making the first electrode layer positively charged and the second electrode layer negatively charged. With the first and second electrode layers charged, an electric field is generated in the cavity formed by the holes in the insulating layer and the surfaces to which the first and second electrode layers are attached. After placing a cell sample in the cavity, the cell sample can be electrochemically converted.

[0048] Furthermore, since the first friction layer rotates at a position a first gap away from the surface of the first electrode layer, and the second friction layer rotates at a position a second gap away from the surface of the second electrode layer, the electric field generated by the first electrode layer and the second electrode layer is stabilized, thus avoiding irreversible damage to the cell membrane during electroconversion treatment, which could lead to low cell survival rate.

[0049] Furthermore, since the layers of the cell electroporator are not glued together, the cell samples inside the chamber can be cleaned by disassembling the cell electroporator, making the operation of the cell electroporator simpler and thus improving the efficiency of cell electroporation to some extent.

[0050] Meanwhile, the cell electroporator has a relatively simple structure, low cost, and very stable performance, which allows the price of the instrument to be controlled at a few yuan to tens of yuan. Compared with the cell electroporators in the existing technology, the cost can be reduced by thousands of times.

[0051] As one embodiment, the first electrode layer 201, the second electrode layer 202, the first friction layer 203, the second friction layer 204, and the insulating layer 205 can be objects of any shape, and there is no specific limitation. For ease of rotation, please refer to... Figure 4 The first electrode layer 201, the second electrode layer 202, the first friction layer 203, the second friction layer 204, and the insulating layer 205 can all be annular cylinders with a central hole. Figure 4 Taking the insulating layer 205 with 6 holes 2051 as an example, the rotating device 206 is connected to the first electrode layer 201, the second electrode layer 202, the first friction layer 203, the second friction layer 204 and the insulating layer 205 through each central hole.

[0052] As one embodiment, both the first electrode layer 201 and the second electrode layer 202 can be circular cylinders with a thickness of 1 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm. A laser cutting machine can be used to cut the rings from a 1 mm thick acrylic sheet to dimensions of 36 mm inner diameter and 70 mm outer diameter. The thickness, inner diameter, and outer diameter of the first electrode layer 201 and the second electrode layer 202 can be set according to specific application requirements and are not limited here.

[0053] Copper foil is adhered to the surface of the insulating layer 205 and the surface adjacent to the first friction layer 203 or the second friction layer 204 on the first electrode layer 201 and the second electrode layer 202. In other words, copper foil is uniformly adhered to both sides of the acrylic ring obtained by laser cutting, thereby completing the fabrication of the first electrode layer 201 and the second electrode layer 202. The first electrode layer 201 and the second electrode layer 202 can be charged by rotating the first friction layer 203 and the second friction layer 204, respectively.

[0054] As one embodiment, both the first friction layer 203 and the second friction layer 204 can be circular cylinders with a thickness of 1 mm, an inner diameter of 24 mm, and an outer diameter of 60 mm. A laser cutting machine can be used to cut the rings from a 1 mm thick acrylic sheet to dimensions of 24 mm inner diameter and 60 mm outer diameter. The thickness, inner diameter, and outer diameter of the first friction layer 203 and the second friction layer 204 can be set according to specific application requirements and are not limited here.

[0055] A nylon film is adhered to the surface of the first friction layer 203 adjacent to the first electrode layer 201, so that the first electrode layer 201 can become positively charged when the first friction layer 203 is rotated. A fluorinated ethylene propylene copolymer (FEP) film is adhered to the surface of the second friction layer 204 adjacent to the second electrode layer 202, so that the second electrode layer 202 can become negatively charged when the second friction layer 204 is rotated. The fabrication of the first friction layer 203 and the second friction layer 204 is thus completed.

[0056] As one embodiment, rabbit hair can be adhered to the surfaces of the first friction layer 203 and the second friction layer 204 adjacent to the first electrode layer 201 or the second electrode layer 202. This allows direct contact between the first friction layer 203 and the first electrode layer 201, and between the second friction layer 204 and the second electrode layer 202, thereby promoting charge transfer and increasing the voltage output. The voltage output can be increased to several thousand to tens of thousands of volts. Therefore, the presence and amount of rabbit hair can be selected according to specific application requirements.

[0057] As one embodiment, the insulating layer 205 can be a cylindrical ring with a thickness in the range of 0.5 to 2 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm, and the inner diameter of the hole 2051 is 6 mm. Within the 0.5 to 2 mm range, at a relatively low rotational speed, the electric field formed between the first electrode layer 201 and the second electrode layer 202 can be suitable for controlling the reversible generation of small pores in the cell membrane, avoiding situations such as consuming excessive driving force or generating an excessively strong electric field. The thickness, inner and outer diameters of the insulating layer, as well as the size and number of holes, can be set according to specific application requirements and are not limited here.

[0058] A laser cutting machine can be used to cut rings into a silicone pad with a thickness of 0.5 to 2 mm, with an inner diameter of 36 mm and an outer diameter of 70 mm. Then, at least one circular hole with an inner diameter of 6 mm is symmetrically cut into the surface of the obtained silicone pad to complete the preparation of the insulating layer.

[0059] As one example, please refer to Figure 5 The fixed component 2061 is a bearing 501, and the rotating component 2062 is a bearing pin 502. The bearing pin 502 is connected to the inner wall of the bearing 501 by a rolling filler 503. The first electrode layer 201, the second electrode layer 202, and the insulating layer 205 are fixed to the outer wall of the bearing 501, and the first friction layer 203 and the second friction layer 204 are fixed to the bearing pin 502. Therefore, since the first electrode layer 201, the second electrode layer 202, and the insulating layer 205 have relatively large masses, while the first friction layer 203 and the second friction layer 204 have relatively small masses, rotating the bearing pin 502 can drive the first friction layer 203 and the second friction layer 204 to rotate, causing the first electrode layer 201 and the second electrode layer 202 to become charged due to the triboelectric effect.

[0060] As one embodiment, the rotating device 206 also includes a pull rope 2063, and the bearing pin 502 is cylindrical. Please refer to [reference needed]. Figure 6 A double-hole 504, symmetrical about the central axis of the cylinder, is provided on the bearing pin 502. Please refer to... Figure 7The pull rope 2063 can pass through the double hole 504. When the pull rope 206 is in a twisted state, it drives the bearing pin 502 to rotate under the action of an external force from the opposite direction. The external force from the opposite direction can be human power or driving force provided by the equipment, etc., and there are no restrictions on the specifics.

[0061] As one embodiment, the rotating device 206 is made of zirconia ceramic material, so that the bearing pin 502 can achieve high-speed rotation without additional lubrication.

[0062] Based on the same inventive concept, embodiments of this application provide a method for cell electroconversion, which uses the aforementioned cell electroconversion instrument to perform cell electroconversion treatment on a cell sample. The cell sample is obtained through at least one chamber. A rotating device is controlled to rotate at a gradually increasing speed, causing a first friction layer and a second friction layer to rotate, thereby creating an electric field between the first electrode layer and the second electrode layer within at least one chamber. When it is determined that charged substances in the solution contained in the cell sample have entered the cell membrane of the cells contained in the cell sample, the rotating device is controlled to stop rotating, and the cell electroconversion result is obtained.

[0063] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A cell electroporator, characterized in that, It includes a first electrode layer, a second electrode layer, a first friction layer, a second friction layer, an insulating layer, and a rotating device, wherein: The first electrode layer and the second electrode layer are respectively attached to different surfaces of the insulating layer. The insulating layer has at least one hole, and the hole, together with the surfaces of the first electrode layer and the second electrode layer, forms a chamber. The chamber is used to contain cell samples. The first friction layer is adjacent to the first electrode layer, and the second friction layer is adjacent to the second electrode layer; The rotating device includes a fixed component and a rotating component. The first electrode layer, the second electrode layer, and the insulating layer are fixed to the fixed component, and the first friction layer and the second friction layer are fixed to the rotating component. The rotating component drives the first friction layer to rotate at a position a first gap away from the surface of the first electrode layer, so that the first electrode layer becomes positively charged. The rotating component drives the second friction layer to rotate at a position a second gap away from the surface of the second electrode layer, so that the second electrode layer becomes negatively charged. After the first electrode layer and the second electrode layer are induced to be charged, an electric field is formed in the cavity.

2. The cell electroporator according to claim 1, characterized in that, The first electrode layer, the second electrode layer, the first friction layer, the second friction layer, and the insulating layer are all annular cylinders with central holes, and the rotating device is connected to the first electrode layer, the second electrode layer, the first friction layer, the second friction layer, and the insulating layer through the central holes.

3. The cell electroporator according to claim 2, characterized in that, The fixed component is a bearing, the rotating component is a bearing pin, the bearing pin is connected to the inner wall of the bearing by a rolling filler, the first electrode layer, the second electrode layer and the insulating layer are fixed to the outer wall of the bearing, and the first friction layer and the second friction layer are fixed to the bearing pin.

4. The cell electroporator according to claim 3, characterized in that, The rotating device also includes a pull rope. The bearing pin is a cylinder with a double hole on the bearing pin, the cylinder having a central axis as the axis of symmetry. The pull rope passes through the double hole. When the pull rope is in a torsional state, it drives the bearing pin to rotate by an external force from the opposite direction.

5. The cell electroporator according to any one of claims 1 to 4, characterized in that, The first electrode layer and the second electrode layer are annular cylinders with a thickness of 1 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm; Copper foil is attached to the surface of the insulating layer and the surface adjacent to the first or second friction layer on the first and second electrode layers.

6. The cell electroporator according to any one of claims 1 to 4, characterized in that, The first friction layer and the second friction layer are annular cylinders with a thickness of 1 mm, an inner diameter of 24 mm, and an outer diameter of 60 mm; A nylon film is adhered to the surface of the first friction layer adjacent to the first electrode layer, and a fluorinated ethylene propylene copolymer film is adhered to the surface of the second friction layer adjacent to the second electrode layer.

7. The cell electroporator according to claim 6, characterized in that, Rabbit fur is attached to the surface of the first friction layer and the second friction layer that is adjacent to the first electrode layer or the second electrode layer.

8. The cell electroporator according to any one of claims 1 to 4, characterized in that, The insulating layer is a cylindrical ring with a thickness of 0.5 to 2 mm, an inner diameter of 36 mm, and an outer diameter of 70 mm. The inner diameter of the hole is 6 mm.

9. The cell electroporator according to any one of claims 1 to 4, characterized in that, The rotating device is made of zirconia ceramic material.

10. A method for cell electroporation, characterized in that, The cell electroporator as described in any one of claims 1 to 9 comprises: Cell samples are obtained through at least one chamber; The rotating device is controlled to rotate at a gradually increasing speed, thereby causing the first friction layer and the second friction layer to rotate, so that the first electrode layer and the second electrode layer form an electric field in the at least one cavity. When it is determined that the charged substance in the solution contained in the cell sample has entered the cell membrane of the cells contained in the cell sample, the rotating device is controlled to stop rotating to obtain the cell electroconversion result.

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