Cell electroporation method and device

Abstract

According to the cell electroporation method and device, an electroporation loading part formed by buckling a first electrode plate and a second electrode plate is arranged, and the number of electrode plates welded to each electrode plate is the same as the number of cell culture pore cavities of a standard cell porous plate; the adjacent first electrode plates and second electrode plates are combined to form a complete positive and negative electrode group, and the positions of the positive and negative electrode groups are in one-to-one correspondence with the cell culture pore cavities, so that a standard cell culture porous plate for bearing a cell suspension can be embedded into the electroporation loading part for direct electroporation without transferring the cell suspension; the non-contact butt joint between the standard cell culture porous plate and the electroporation loading part is realized, the damage of electroporation to cells is smaller, the cell survival rate is improved, a cell porous plate of a special specification does not need to be customized, plug-and-play is realized, the problem that an existing electroporation instrument is not matched with the standard cell culture porous plate is solved, the cost is reduced, the operation process is simplified, and the operation efficiency is improved. And the high-flux electrotransfection is realized.

Description

Technical Field

[0001] The present application relates to the field of cell biological experiment technology, and in particular to a cell electroporation method and device. Background Art

[0002] Intracellular delivery, or cell transfection, is a critical step in biological research and has enabled decades of biomedical discoveries. Intracellular delivery technology is also becoming increasingly important in industrial and medical applications ranging from biomanufacturing to cell-based therapies.

[0003] The main challenge of intracellular delivery technology is to introduce membrane-impermeable drugs into the interior of cells without causing adverse damage to the cells. In intracellular delivery technology, electroporation is a commonly used method.

[0004] Electroporation technology uses the principle that when an external electric field is applied to biological cells, the lipid bimolecules on the cell membrane are reversed to form hydrophilic micropores. An external electric field is applied to biological cells. If the cell membrane can return to normal after the external electric field is removed, drugs can be introduced into the cells or components in the cells can be extracted.

[0005] Traditional electroporation devices have been widely used. The general process is: pipette the cell suspension into a culture dish for culture. After culture, pipette the mixture into a cuvette, set the voltage and capacitance, and insert the cuvette into the electroporator for electroporation.

[0006] However, the traditional electroporation device and the corresponding electroporation process have a major flaw: they cannot be adapted to the standard cell culture multi-well plates specified by existing international standards, and the cultured cell suspension to be electroporated needs to be transferred, which can lead to problems such as cell death, sample contamination, and difficulty in improving efficiency.

[0007] This is because traditional electroporation devices and the corresponding electroporation processes require the cell suspension to be transferred to the electroporation instruments of their respective systems, which increases the difficulty of experimental operations. In the transfer process, it will cause cell death, increase the probability of sample contamination, and increase the difficulty of manual operation. At the same time, in the traditional electrode mode, the electrode is in contact with the cell electroporation solution, and a large amount of metal ions are generated during the electrolysis process, causing a large number of cells to die, resulting in low electroporation efficiency. Summary of the invention

[0008] Based on this, it is necessary to provide a cell electroporation device to address the problems that traditional cell electroporation methods and devices cannot be adapted to the standard cell culture multi-well plates specified by existing international standards, and the cultured cell suspension to be electroporated needs to be transferred, which may lead to cell death, sample contamination, and difficulty in improving efficiency.

[0009] The present application provides a cell electroporation device, which is used in conjunction with a standard cell culture multi-well plate, and the cell electroporation device comprises:

[0010] The electroporation loading part is embedded in a standard cell culture multi-well plate; the bottom surface of the standard cell culture multi-well plate is provided with M cell culture cavities arranged in a matrix, and the cell culture cavities are used to carry a cell suspension; M is a positive integer and M is greater than or equal to 4;

[0011] An electrical signal generating unit, used for generating an electrical signal and applying the electrical signal to the electroporation loading unit;

[0012] The electroporation loading unit comprises:

[0013] A first electrode plate, the first electrode plate being welded with M first electrode sheets arranged in a matrix;

[0014] A second electrode plate, the second electrode plate being welded with M second electrode sheets arranged in a matrix;

[0015] One of the first electrode sheet and the second electrode sheet is a positive electrode sheet, and the other is a negative electrode sheet;

[0016] The first electrode plate and the second electrode plate are matched and buckled with each other, so that the first electrode plate and the second electrode plate that are closest to each other form a positive and negative electrode group with an internal accommodation space, so that the entire electroporation loading part has M positive and negative electrode groups;

[0017] When the standard cell culture multi-well plate is embedded in the electroporation loading part, each cell culture cavity is embedded in a receiving space of a positive and negative electrode group aligned with the position of the cell culture cavity, so that the electric signal applied by the positive and negative electrode group forms an electric field in the receiving space, and applies the electric field to the cell culture cavity that is aligned with the positive and negative electrode group.

[0018] The present application also provides a cell electroporation method, which is applied to the cell electroporation device mentioned above, and the cell electroporation method comprises:

[0019] A portion of the cell suspension is placed in a cell culture well chamber of a standard cell culture multi-well plate, and a transfection substance is added to the cell culture well chamber;

[0020] Repeat the steps of placing a portion of the cell suspension in a cell culture well cavity in a standard cell culture multi-well plate, and adding a transfection substance to the cell culture well cavity, until the cell suspension and the transfection substance are added to each of the cell culture well cavities in the target number of cell culture well cavities;

[0021] Insert a standard cell culture multi-well plate into the electroporation loading section;

[0022] Set electroporation parameters;

[0023] Driving the electric signal generating unit to generate an electric signal and applying the electric signal to the electroporation loading unit;

[0024] Take out the standard cell culture multiwell plate.

[0025] The present application relates to a cell electroporation method and device, which is provided with an electroporation loading part formed by interlocking a first electrode plate and a second electrode plate, and the number of electrode sheets welded on each electrode plate is the same as the number of cell culture cavities of a standard cell culture porous plate, and the adjacent first electrode sheet and the second electrode sheet are combined to form a complete positive and negative electrode group, and the position of the positive and negative electrode group corresponds to the cell culture cavities one by one, so that the standard cell culture porous plate carrying a pre-cultured cell suspension can be directly electroporated by embedding it in the electroporation loading part, without the need to transfer the cell suspension, so as to achieve contactless docking between the standard cell culture porous plate and the electroporation loading part, so that the electroporation process causes less damage to cells and improves cell survival rate. In addition, the present application directly uses a cell culture porous plate that meets the standard, and there is no need to specially customize a cell culture porous plate of special size specifications and other redundant equipment. It is plug-and-play, and the standard cell culture porous plate can be pulled out after the electroporation is completed, which solves the problem of incompatibility between the current electroporation instrument and the cell culture porous plate that meets the standard, reduces costs, and simplifies the operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the structure of a cell electroporation device provided in one embodiment of the present application.

[0027] Figure 2 Schematic diagram of the connection relationship between the first electrode plate, the first switch, the second electrode plate and the second switch in the cell electroporation device provided in one embodiment of the present application.

[0028] Figure 3 Schematic diagram of the structure of a standard cell culture multi-well plate in a cell electroporation device provided in one embodiment of the present application.

[0029] Figure 4 Schematic diagram of the structure of the second electrode plate in the cell electroporation device provided in one embodiment of the present application.

[0030] Figure 5 Schematic diagram of the structure of the first electrode plate in the cell electroporation device provided in one embodiment of the present application.

[0031] Figure 6 Schematic diagram of the structure of a support in a cell electroporation device provided in one embodiment of the present application.

[0032] Figure 7FIG. 1 is a schematic diagram of a cell electroporation device provided in an embodiment of the present application after being assembled with a standard cell culture multi-well plate.

[0033] Figure 8 For Figure 7 The structure shown is a half-section view taken along section line AA'.

[0034] Fig. 9 Schematic diagram of the connection relationship between the first electrode plate, the second electrode plate and the bracket in the cell electroporation device provided in one embodiment of the present application.

[0035] Fig.10 For Fig. 9 A magnified schematic diagram of point B in the middle.

[0036] Fig.11 Schematic diagram of the assembly of a standard cell culture multi-well plate, a first electrode plate, a second electrode plate and a bracket of a cell electroporation device provided in one embodiment of the present application.

[0037] Fig.12 1 is a flow chart of a cell electroporation method provided in one embodiment of the present application.

[0038] Fig.13 is a circuit diagram of an early warning circuit in a cell electroporation device provided in one embodiment of the present application.

[0039] Fig.14 1 is a circuit diagram of a circuit coupling a test circuit and a perforation electrical signal generating circuit in a cell electroporation device provided in an embodiment of the present application.

[0040] Fig.15 1 is an electric field simulation diagram of a cell electroporation device provided in an embodiment of the present application applying an electric field to a single cell culture cavity at 100 V, 300 V, 500 V, 800 V, 1000 V and 1200 V respectively.

[0041] Fig.16 1 is a comparison curve diagram of electric field uniformity when the cell electroporation device provided in one embodiment of the present application applies an electric field at 100V, 300V, 500V, 800V, 1000V and 1200V in a single cell culture cavity.

[0042] Fig.17 The electric field bright field image after applying an electric field to a single cell culture cavity using the cell electroporation device and method provided in one embodiment of the present application and the fluorescence image after electrotransfection, as well as the electric field bright field image after applying an electric field to a single cell culture cavity using a traditional cell electroporation device and method and the fluorescence image after electrotransfection.

[0043] Reference numerals:

[0044] 10-standard cell culture multi-well plate; 11-cell culture cavity; 100-electroporation loading part;

[0045] 110 - first electrode plate; 111 - first electrode sheet; 112 - first through hole; 120 - second electrode plate;

[0046] 121 - second electrode sheet; 130 - accommodation space; 140 - first elastic mechanism; 141 - first wedge block;

[0047] 142 - first spring; 150 - second elastic mechanism; 151 - second wedge block; 152 - second spring;

[0048] 200-electrical signal generating unit; 210-main control and signal generating unit; 220-high voltage generating unit; 300-support;

[0049] 310 - second through hole; 320 - first handle; 330 - second handle; 400 - positive and negative electrode group;

[0050] 500- switch assembly; 510- first switch; 520- second switch; 600- energy supply unit;

[0051] 700-human-computer interaction and display unit; 800-base; 810-storage slot. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solution and advantages of this application more clear, the following is a further detailed description of this application in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain this application and are not used to limit this application.

[0053] The present application provides a cell electroporation device. It should be noted that the cell electroporation device provided in the present application is applied to a standard cell culture multi-well plate designed according to international cell multi-well plate standards.

[0054] like Figure 1 As shown, in one embodiment of the present application, the cell electroporation device is used in conjunction with a standard cell culture multi-well plate 10. The standard cell culture multi-well plate 10 is a standard cell culture multi-well plate designed according to international cell multi-well plate standards.

[0055] like Figure 1 As shown, the cell electroporation device includes an electroporation loading unit 100 and an electrical signal generating unit 200 .

[0056] The electroporation loading unit 100 is inserted into a standard cell culture multi-well plate 10. Figure 3As shown, the bottom surface of the standard cell culture multi-well plate 10 is provided with M matrix-arranged cell culture cavities 11, and the cell culture cavities 11 are used to carry the cell suspension. M is a positive integer and M is greater than or equal to 4. The electrical signal generating unit 200 is used to generate an electrical signal and apply the electrical signal to the electroporation loading unit 100.

[0057] like Figure 2 As shown, the electroporation loading unit 100 includes a first electrode plate 110 and a second electrode plate 120. Figure 5 As shown, the first electrode plate 110 is welded with M first electrode sheets 111 arranged in a matrix. Figure 4 As shown, the second electrode plate 120 is welded with M second electrode sheets 121 arranged in a matrix. One of the first electrode sheet 111 and the second electrode sheet 121 is a positive electrode sheet, and the other is a negative electrode sheet.

[0058] like Figure 8 As shown, the first electrode plate 110 and the second electrode plate 120 match and snap together, so that the first electrode sheet 111 and the second electrode sheet 121 that are closest to each other form a positive and negative electrode group 400 with an internal accommodation space 130, so that the entire electroporation loading part 100 has M positive and negative electrode groups 400.

[0059] When the standard cell culture multi-well plate 10 is embedded in the electroporation loading part 100, each cell culture cavity 11 is embedded in a receiving space 130 of a positive and negative electrode group 400 aligned with the position of the cell culture cavity 11, so that the electric signal applied by the positive and negative electrode group 400 forms an electric field in the receiving space 130 of the positive and negative electrode group 400, and applies the electric field to the cell culture cavity 11 corresponding to the positive and negative electrode group 400.

[0060] Specifically, one of the first electrode sheet 111 and the second electrode sheet 121 is a positive electrode sheet, and the other electrode sheet is a negative electrode sheet, that is, the first electrode sheet 111 can be a positive electrode sheet, and the second electrode sheet 121 can be a negative electrode sheet. In this embodiment, the first electrode plate 110 is a positive electrode plate, and all the positive electrode sheets are arranged thereon, and the second electrode plate 120 is a negative electrode plate, and all the negative electrode sheets are arranged thereon.

[0061] The first electrode sheet 111 can also be a negative electrode sheet, and the second electrode sheet 121 is a positive electrode sheet. In this embodiment, the first electrode plate 110 is a negative electrode plate, and all the negative electrode sheets are arranged on it. The second positive electrode plate is a positive electrode plate, and all the positive electrode sheets are arranged on it.

[0062] A positive electrode sheet and a negative electrode sheet form a complete positive and negative electrode group 400, such as Figure 8 In a positive and negative electrode group 400, the positive electrode sheet generates a positive electrical signal, the negative electrode sheet generates a negative electrical signal, and the entire positive and negative electrode group generates a periodic arbitrary wave, which can be a sine wave or a square wave.

[0063] It should be noted that the standard cell culture multi-well plate 10 used in this embodiment is designed according to international cell multi-well plate standards, and the current standard cell culture multi-well plate 10 that meets the standards can be directly used as a container to implement the electroporation process.

[0064] Alternatively, if Figure 1 As shown, the cell electroporation device further includes an energy supply unit 600 and a human-computer interaction and display unit 700 .

[0065] The electrical signal generating unit 200 includes a main control and signal generating unit 210 and a high voltage generating unit 220 .

[0066] The energy supply unit 600 is electrically connected to the human-machine interaction and display unit 700, and is used to supply power to the human-machine interaction and display unit 700. The energy supply unit 600 is electrically connected to the main control and signal generating unit 210, and is used to supply power to the main control and signal generating unit 210. The energy supply unit 600 is electrically connected to the high voltage generating unit 220, and is used to supply power to the high voltage generating unit 220.

[0067] The main control and signal generating unit 210 is directly connected to the high voltage generating module to form a perforation electrical signal generating circuit.

[0068] The high voltage generating unit 220 is electrically connected to the electroporation loading unit 100 .

[0069] The main control and signal generating unit 210 is electrically connected to the human-machine interaction and display unit 700 , and the operator can select the working mode, set the electroporation parameters, and display the real-time information of the electroporation process on the human-machine interaction and display unit 700 .

[0070] When in use, a cell culture multi-well plate 10 that meets the standards is embedded in the electroporation loading unit 100, and the working mode of the entire cell electroporation device is selected through the human-computer interaction and display unit 700. An electrical signal is generated in the perforation electrical signal generating circuit, and the electrical signal acts on the electroporation loading unit 100 to complete the electroporation process of the cells in the cell culture multi-well plate 10 that meets the standards.

[0071] In this embodiment, an electroporation loading part 100 is provided, which is formed by a first electrode plate 110 and a second electrode plate 120 that are interlocked with each other. The number of electrode sheets welded on each electrode plate is set to be the same as the number of cell culture cavities 11 of the standard cell culture multi-well plate 10, and the adjacent first electrode sheets 111 and second electrode sheets 121 are combined to form a complete electrode, and the electrode positions correspond to the cell culture cavities 11 one by one, so that the standard cell culture multi-well plate 10 carrying the pre-cultured cell suspension can be embedded in the electroporation loading part 100 to directly perform the electroporation operation without transferring the cell suspension, thereby realizing contactless docking between the standard cell culture multi-well plate 10 and the electroporation loading part 100, so that the electroporation process causes less damage to the cells and improves the cell survival rate. In addition, the present application directly uses a standard cell culture multi-well plate 10, and there is no need to specially customize a cell culture multi-well plate of special size and specifications and other redundant equipment. It is plug and play, and the cell culture multi-well plate can be pulled out after the electroporation is completed. This solves the problem of incompatibility between the current electroporation instrument and the standard cell culture multi-well plate 10, reduces costs, and simplifies the operation process.

[0072] In addition, in the present embodiment, the first electrode sheet 111 on the first electrode plate 110 adopts a matrix electrode sheet layout that is the same as the cell culture cavity 11 layout of the standard cell porous plate 10, and the second electrode sheet 121 on the second electrode plate 120 also adopts a matrix electrode sheet layout that is the same as the cell culture cavity 11 layout of the standard cell porous plate 10. The standard cell porous plate 10 is directly used as an electroporation container to complete the cell transfection process without contacting the cell suspension, which not only eliminates the need to specially customize cell porous plates of special sizes and other redundant equipment, that is, not only does it not require customized exclusive cell culture porous plates, but it also eliminates the need for electroporation cups and electroporation fluids.

[0073] In addition, the cell electroporation device provided in this embodiment can realize high-throughput electroporation transfection work and can perform electroporation operations at multiple cell culture cavity locations simultaneously.

[0074] like Figure 4 and Figure 5 As shown, in one embodiment of the present application, the first electrode sheet 111 and the second electrode sheet 121 have the same structure and the same size, the first electrode sheet 111 and the second electrode sheet 121 are symmetrically arranged, and the positive and negative electrode group 400 composed of the first electrode sheet 111 and the second electrode sheet 121 is arranged in a surrounding manner around the cell culture cavity 111, as shown in FIG. Figure 8 The cell culture cavity 11 is a cylindrical shell with a hollow interior. The heights of the first electrode sheet and the second electrode sheet are both greater than the height of the cell culture cavity.

[0075] Specifically, in this embodiment, the first electrode sheet 111 and the second electrode sheet 121 form a positive and negative electrode group 400. The positive and negative electrode group 400 is hollow and has a receiving space 130. This is an insertion electrode with a semi-enclosed structure, which can be inserted into the cell culture cavity 11 of the standard cell culture multi-well plate 10 to realize the electroporation process.

[0076] The cell culture cavity 11 is a cylindrical shell with a hollow interior, which may be an open top, closed bottom, and a hollow cup-shaped structure inside, and is used to accommodate a sufficient amount of cell suspension for the experiment and the subsequent addition of electro-transfection substances.

[0077] In an optional implementation, the first electrode sheet 111 is an arc-shaped electrode sheet. The second electrode sheet 121 is an arc-shaped electrode sheet. Figure 4 and Figure 5 As shown, the first electrode sheet 111 and the second electrode sheet 121 are symmetrically arranged so that the positive and negative electrode group 400 composed of the first electrode sheet 111 and the second electrode sheet 121 is in a bracket shape.

[0078] The heights of the first electrode sheet and the second electrode sheet are both greater than the height of the cell culture cavity. Fig. 9 The Z axis is perpendicular to the first electrode plate 110 and the second electrode plate 120 .

[0079] In this embodiment, by arranging the first electrode sheet 111 and the second electrode sheet 121 symmetrically and the positive and negative electrode group 400 composed of the first electrode sheet 111 and the second electrode sheet 121 is surrounded on all sides of the cell culture cavity 11, the inserted electrode of the electrode semi-enclosed structure composed of the first electrode sheet 111 and the second electrode sheet 121 can wrap the single cell culture cavity 11 after the first electrode sheet 111 and the second electrode sheet 121 are embedded in the standard cell culture porous plate 10, thereby achieving direct docking and adaptation of the entire cell electroporation device and the standard cell culture porous plate 10, simplifying the experimental process, avoiding unnecessary cell death under mechanical stress conditions, and improving the electrotransfection flux. At the same time, while maintaining the electric field, it also avoids the problem of cell death caused by electrode electrolysis.

[0080] like Figure 5 As shown, in one embodiment of the present application, the first electrode plate 110 is provided with a plurality of first through holes 112 , the outer diameter of the cell culture cavity 11 is smaller than the diameter of the first through hole 112 , and the outer diameter of the positive and negative electrode group 400 is smaller than the diameter of the first through hole 112 .

[0081] Specifically, the outer diameter of the cell culture cavity 11 is the diameter of the circular bottom surface of the cylindrical cell culture cavity 11. The outer diameter of the positive and negative electrode group 400 is the diameter of the equivalent circular bottom surface of the cylindrical structure formed by the positive and negative electrode group 400.

[0082] In this embodiment, the outer diameter of the cell culture cavity 11 is smaller than the diameter of the first through hole 112, which is conducive to inserting the cell culture cavity 11 into the first through hole 112 to complete the embedding of the standard cell culture multi-well plate 10 into the electroporation loading part 100. The outer diameter of the positive and negative electrode group 400 is smaller than the diameter of the first through hole 112, which is conducive to the positive and negative electrode group 400 being limited in the first through hole 112, and the entire positive and negative electrode group 400 will not shake to cause positioning failure, so that the position of the electrical signal application is more accurate.

[0083] Optionally, in one embodiment of the present application, the top of each first electrode sheet 111 on the first electrode plate 110 abuts against the inner hole wall of the first through hole 112, and the top of the first electrode sheet 111 may be flush with the first through hole 112 or slightly higher than the first through hole 112. The first electrode sheet 111 extends from the bottom surface of the first electrode plate 110.

[0084] Each electrode sheet on the second electrode plate 120 is disposed on the top surface of the second electrode plate 120 .

[0085] This embodiment realizes a novel double-layer mesh structure circuit board design, in which the first electrode sheet 111 is on the bottom surface of the first electrode plate 110, and the second electrode sheet 121 is on the top surface of the second electrode plate 120. When the first electrode plate 110 and the second electrode plate 120 are buckled together, the first electrode sheet 111 and the second electrode sheet 121 form a positive and negative electrode group 400, which can prevent the electric fields formed by adjacent electrodes from interfering with each other. In addition, this method saves layout space, increases the number of holes that can be electroporated, and improves the flux of electrotransfection.

[0086] like Figure 6 As shown, in one embodiment of the present application, the cell electroporation device further includes a support 300 .

[0087] The bracket 300 is configured as a plate-like structure, and the bracket 300 is provided with M second through holes 310 arranged in a matrix. Figure 6 As shown, a first handle 320 in the shape of a slat is disposed on one side of the bracket 300 , and a second handle 330 in the shape of a slat is disposed on the other side of the bracket 300 .

[0088] The M first through holes 112 and the M second through holes 310 are arranged in a one-to-one alignment, that is, each first through hole 112 has a unique second through hole 310 aligned with its position.

[0089] like Figure 8 As shown, when the standard cell culture multi-well plate 10 is embedded in the electroporation loading part 100, the standard cell culture multi-well plate 10 is first interlocked with the bracket 300 to form an assembly, and the assembly is then embedded in the electroporation loading part 100 to complete the embedding of the standard cell culture multi-well plate 10 in the electroporation loading part 100.

[0090] Specifically, the two handles of the bracket 300 are as follows: Figure 6 and Figure 7 shown.

[0091] In this embodiment, the setting of the bracket 300 can make it easy to insert and remove the standard cell culture multi-well plate 10 from the electroporation loading part 100. The standard multi-well plate can be snapped together with the bracket 300 to complete a preliminary installation to form an assembly. At this time, the entire assembly can be easily plugged in and out of the electroporation loading part 100 through the handle, which will not damage the cells in the standard cell culture multi-well plate 10 and has the function of stabilizing the position of the positive and negative electrode groups 400.

[0092] In one embodiment of the present application, the outer diameter of the cell culture cavity 11 is smaller than the diameter of the second through hole 310 .

[0093] In this embodiment, the outer diameter of the cell culture cavity 11 is smaller than the diameter of the second through hole 310 , so that the cell culture cavity 11 and the second through hole 310 can be interference-fitted.

[0094] like Figure 4 and Figure 5 As shown, in one embodiment of the present application, the electroporation loading unit 100 further includes a plurality of first elastic mechanisms 140 and a plurality of second elastic mechanisms 150 .

[0095] Each first electrode sheet 111 is provided with a first elastic mechanism 140, a portion of which is embedded in the first electrode sheet 111, and another portion thereof is extended out of the outer circumference of the first electrode sheet 111. Each second electrode sheet 121 is provided with a second elastic mechanism 150. A portion of the second elastic mechanism 150 is embedded in the second electrode sheet 121, and another portion thereof is extended out of the outer circumference of the second electrode sheet 121. The outer diameter of the positive and negative electrode groups 400 is smaller than the diameter of the second through hole 310.

[0096] Specifically, a fixing mode and a clamping mode may be used. When the clamping mode is switched, the first electrode sheet 111 and the second electrode sheet 121 may generate the following Fig. 9 As shown in the displacement in the X-axis direction, the first electrode sheet 111 and the second electrode sheet 121 will be closely attached to the cell culture cavity 11 on the standard multi-well plate.

[0097] When we want to enter clamping mode:

[0098] By pressing the assembly downward with the handle, when the assembly is pressed downward, the first elastic mechanism 140 and the second elastic mechanism 150 both move downward into the second through hole 310, the first elastic mechanism 140 retracts under the action of elasticity and drives the first electrode sheet 111 to move toward the direction close to the cell culture cavity 11, and the second elastic mechanism 150 retracts under the action of elasticity and drives the second electrode sheet 121 to move toward the direction close to the cell culture cavity 11, so that the first electrode sheet 111 and the second electrode sheet 121 clamp the cell culture cavity 11. The portion of the first elastic mechanism 140 extending out of the outer circumference of the first electrode sheet 111 abuts against the inner hole wall of the second through hole 310, and the portion of the second elastic mechanism 150 extending out of the outer circumference of the second electrode sheet 121 abuts against the inner hole wall of the second through hole 310 to maintain the clamping state.

[0099] When we want to get out of the clamping mode and change to the loosening mode, and then take out the assembly:

[0100] By lifting the assembly by the handle, when the assembly is lifted, the first elastic mechanism 140 and the second elastic mechanism 150 are both moved upward and separated from the second through hole 310, the first elastic mechanism 140 expands outward under the action of elasticity and drives the first electrode sheet 111 to move away from the cell culture cavity 11, and the second elastic mechanism 150 expands outward under the action of elasticity and drives the second electrode sheet 121 to move away from the cell culture cavity 11, so that the first electrode sheet 111 and the second electrode sheet 121 no longer clamp the cell culture cavity 11. At this time, the assembly can be taken out, and then the bracket 300 and the standard cell culture multi-well plate 10 are separated, and the next standard cell culture multi-well plate 10 is replaced to prepare for the next experiment.

[0101] The outer diameter of the positive and negative electrode group 400 is smaller than the diameter of the second through hole 310 because the present embodiment needs to introduce a clamping mode, so the positive and negative electrode group 400 must enter the second through hole 310. The aforementioned embodiment can also implement the present application without introducing a clamping mode, and the outer diameter of the positive and negative electrode group 400 is not forced to be smaller than the diameter of the second through hole 310, because the top of the positive and negative electrode group 400 can be against the bottom of the bracket 300, and it is not necessary to move up to enter the second through hole 310.

[0102] In this embodiment, a first elastic mechanism 140 is arranged on the first electrode sheet 111, and a second elastic mechanism 150 is arranged on the second electrode sheet 121, so as to form a microscale clamp electrode. When in a clamped state, the air gap is reduced, which is conducive to maintaining a high-intensity uniform electric field and improving the accuracy and safety of electric field application.

[0103] Alternatively, if Fig.10As shown, in one embodiment of the present application, the first elastic mechanism 140 includes a first wedge 141 and a first spring 142. As shown, a groove is provided at the top of the first electrode sheet 111 (the groove is not numbered to avoid contradictions in the marking), a portion of the first wedge 141 is inserted into the groove, and the other portion extends out of the first electrode sheet 111, and the portion inserted into the groove is fixedly connected to the first spring 142. One end of the first spring 142 is fixedly connected to the bottom of the groove, and the other end of the first spring 142 is fixedly connected to the first wedge 141. The surface of the portion of the first wedge 141 outputting the first electrode sheet 111 close to the second through hole 310 is a curved surface, so that when the entire first elastic mechanism 140 is retracted in the clamping mode, the portion of the first wedge 141 outputting the first electrode sheet 111 can better abut against the wall surface of the inner hole wall of the second through hole 310, and the clamping state can be maintained through this abutting relationship.

[0104] When entering the clamping mode, the assembly is pressed down through the two handles. When the assembly is pressed down, the first elastic mechanism 140 moves downward as a whole, the first wedge block 141 enters the second through hole 310, and the first wedge block 141 shrinks inward, compressing the first spring 142, so that the first spring 142 is in a compressed state. The part of the first wedge block 141 extending out of the first electrode sheet 111 rests on the wall surface of the inner hole wall of the second through hole 310, clamping the cell culture cavity 11.

[0105] When switching from the clamping mode to the relaxing mode, the assembly is moved upward by the two handles. When the assembly is moved upward, the first elastic mechanism 140 moves upward as a whole, the first wedge block 141 disengages from the second through hole 310, and the first spring 142 stretches, thereby driving the first wedge block 141 to expand outward, thereby loosening the clamping of the cell culture cavity 11.

[0106] like Fig.10 As shown, the second elastic mechanism 150 includes a second wedge block 151 and a second spring 152, and the working principle is the same as that of the first wedge block 141 and the first spring 142, which will not be repeated here.

[0107] When entering the clamping mode, the assembly is pressed down by the two handles. When the assembly is pressed down, the first elastic mechanism 140 and the second elastic mechanism 150 move downward as a whole at the same time, the first wedge block 141 and the second wedge block 151 enter the second through hole 310 at the same time, the first wedge block 141 and the second wedge block 151 retract at the same time, the first wedge block 141 presses the first spring 142, so that the first spring 142 is in a compressed state, the second wedge block 151 presses the second spring 152, so that the second spring 152 is in a compressed state, the part of the first wedge block 141 extending out of the first electrode sheet 111 abuts against the wall surface of the inner hole wall of the second through hole 310, the part of the second wedge block 151 extending out of the second electrode sheet 121 abuts against the wall surface of the inner hole wall of the second through hole 310, and the cell culture cavity 11 is clamped.

[0108] When you want to switch from the clamping mode to the relaxation mode, move the assembly upward through the two handles. When the assembly moves upward, the first elastic mechanism 140 and the second elastic mechanism 150 move upward as a whole at the same time, the first wedge block 141 and the second wedge block 151 are disengaged from the second through hole 310 at the same time, and the first spring 142 and the second spring 152 are stretched at the same time, and then the first spring 142 drives the first wedge block 141 to expand outward, and the second spring 152 drives the second wedge block 151 to expand outward, thereby loosening the clamping of the cell culture cavity 11.

[0109] Alternatively, if Figure 7 and Figure 8 As shown, in one embodiment of the present application, the cell electroporation device further includes a base 800. The base 800 is a box body with an open top and a cubic space inside. The base 800 is used to accommodate the electroporation loading part 100, the standard cell culture multi-well plate 10 and the bracket 300. That is, the cubic space of the base 800 is used to accommodate the electroporation loading part 100, the standard cell multi-well plate 10 and the bracket 300.

[0110] Before the experiment begins, the first electrode plate 110 and the second electrode plate 120 are placed in the base 800 and are buckled with each other.

[0111] After the experiment begins, the standard cell culture multi-well plate 10 and the support 300 are interlocked to form an assembly, and then the assembly is embedded in the base 800 and then enters the clamping mode, finally forming a complete overall structure that can wait for electroporation.

[0112] In order to avoid excessive shaking of the first electrode plate 110 and the second electrode plate 120 in the base 800 before and after clamping, the length of the internal cubic space of the base 800 on the X-axis can be set to be greater than the length of the first electrode plate 110 on the X-axis, and the length of the internal cubic space of the base 800 on the X-axis is also greater than the length of the second electrode plate 120 on the X-axis. Specifically, the difference between the length of the internal cubic space of the base 800 on the X-axis and the length of the first electrode plate 110 can be freely set. For example, the spacing between the first electrode plate 110 and the cell culture cavity 11 in the relaxation mode can be set to 1 mm, and the spacing between the second electrode plate 120 and the cell culture cavity 11 can also be set to 1 mm, then the length of the internal cubic space of the base 800 on the X-axis can be set according to these two spacings.

[0113] The length of the cubic space inside the base 800 on the Y axis is just equal to or slightly greater than the length of the first electrode plate 110 and the second electrode plate 120 on the Y axis, so that redundant space is provided in the X axis direction for the displacement of the first electrode plate 110 and the second electrode plate 120 due to the X axis clamping, but the first electrode plate 110 and the second electrode plate 120 are kept from displacement in the Y axis direction as much as possible. Fig. 9 shown.

[0114] In addition, a receiving groove 810 is respectively formed on two sides of the top of the base 800 to accommodate the first handle 320 and the second handle 330 .

[0115] like Figure 2 As shown, in one embodiment of the present application, the cell electroporation device further includes a switch component 500. The switch component 500 is electrically connected to the main control and signal generating unit 210.

[0116] The switch assembly 500 includes a plurality of first switches 510 and a plurality of second switches 520. The plurality of first switches 510 are disposed on the first electrode plate 110. Each first switch 510 is electrically connected to the main control and signal generating unit 210. Different first switches 510 are electrically connected to different columns of first electrode sheets 111.

[0117] A plurality of second switches 520 are disposed on the second electrode plate 120 , each second switch 520 is electrically connected to the main control and signal generating unit 210 , and different second switches 520 are electrically connected to the second electrode sheets 121 in different rows.

[0118] Specifically, each first switch 510 is electrically connected to the main control and signal generating unit 210. Each second switch 520 is electrically connected to the main control and signal generating unit 210.

[0119] The positions of different cell culture wells 11 of the standard cell culture multi-well plate 10 can be arbitrarily selected to apply electric signals with different waveforms and parameters, that is, by turning on different combinations of the first switch 510 and the second switch 520, electric signals can be applied to the positions of different cell culture wells 11 at any position. Moreover, the parameters of the electric signal can be set, so not only can the cell culture well 11 to which the electric signal is applied be customized, but also the properties of the electric signal itself can be customized, and electric signals with different waveforms and different parameters can be applied.

[0120] The number of first switches 510 multiplied by the number of second switches 520 equals M. In this embodiment, different first switches 510 are electrically connected to different columns of first electrode sheets 111. If the electrode plate has 6 columns of electrode sheets, the number of first switches 510 is 6. In this embodiment, different second switches 520 are electrically connected to different rows of second electrode sheets 121. If the electrode plate has 8 rows of electrode sheets, the number of second switches 520 is 8. Thus, the electric signal application of the position of the custom cell culture cavity 11 is formed by the cooperation of the first switch 510 and the second switch 520.

[0121] It should be noted that, in this embodiment, a first switch 510 and a second switch 520 must be turned on at the same time in order to apply an electrical signal to a cell culture cavity 11 .

[0122] For example, the first switch 510 is A1, A2, A3, A4, A5, A6, and the second switch 520 is B1, B2, B3, B4, B5, B6, B7, B8. If you want to apply an electrical signal to the cell culture cavity 11 in the first row and first column, then turn on the A1 switch and the B1 switch at the same time, and keep the other switches in the closed state.

[0123] In this embodiment, by setting a plurality of first switches 510 and a plurality of second switches 520, it is possible to realize applying an electrical signal to a cell well at any position by turning on different combinations of the first switches 510 and the second switches 520, and it is possible to realize electroporation of different waveforms and different electrical signals at any position of the cell culture well 11 on the standard cell multi-well plate 10.

[0124] like Fig.12 As shown, the present application also provides a cell electroporation method. The cell electroporation method is applied to the cell electroporation device mentioned in any one of the above embodiments.

[0125] The cell electroporation method provided in the present application does not limit its execution subject. Optionally, the execution subject of the cell electroporation method provided in the present application can be the cell electroporation device mentioned in any of the aforementioned embodiments. Specifically, the execution subject of the cell electroporation device provided in the present application can be the main control and signal generating unit 210 in the cell electroporation device mentioned in any of the aforementioned embodiments.

[0126] like Fig.12 As shown, the cell electroporation method comprises the following steps:

[0127] S100 , placing a portion of the cell suspension in a cell culture cavity 11 in a standard cell culture multi-well plate 10 , and adding a transfection substance into the cell culture cavity 11 .

[0128] S200, repeatedly performing S100 until the addition of the cell suspension and the transfection substance is completed in each of the target number of cell culture cavities 11, that is, repeatedly performing the steps of placing a portion of the cell suspension in a cell culture cavity 11 in the standard cell culture multi-well plate 10, and adding the transfection substance to the cell culture cavity 11, until the addition of the cell suspension and the transfection substance is completed in each of the target number of cell culture cavities 11.

[0129] S300 , inserting the standard cell culture multi-well plate 10 into the electroporation loading part 100 .

[0130] S400, setting electroporation parameters.

[0131] S700 , driving the electrical signal generating unit 200 to generate an electrical signal and applying the electrical signal to the electroporation loading unit 100 .

[0132] S900, take out 10 standard cell culture multi-well plates.

[0133] Specifically, S100 includes:

[0134] S110, harvesting an appropriate amount of cells, dispersing and resuspending the cells in a cell culture multi-well plate 10 to form a cell suspension.

[0135] Resuspension means "re-suspending". The specific operation is to re-suspend the solids (precipitates, cells, active substances, etc.) obtained by centrifugation or sedimentation with an appropriate buffer or culture medium.

[0136] S120, adding the desired transfection material into the cell culture multi-well plate 10, so that the cell suspension and the drug to be delivered for electroporation are completely mixed.

[0137] Transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of exogenous DNA.

[0138] S400 includes:

[0139] S410 , starting the human-machine interaction unit and the display unit 700 , and selecting the position of the cell culture cavity 11 to be loaded with the electrical signal through the touch screen of the human-machine interaction unit and the display unit 700 .

[0140] S420 , selecting the desired output electrical signal waveform for each cell culture cavity 11 , and setting parameters for each cell culture cavity 11 .

[0141] The frequency of the applied electrical signal is within a frequency range of greater than 1 Hz and less than 225 Hz, and the peak voltage is within a voltage range of greater than 0 and less than 3000V.

[0142] When S700 is executed, the human-machine interaction unit and the display unit 700 can display the status of electroporation in real time, and confirm whether the electroporation is successful. In this application, electroporation is equivalent to electroporation, and will not be described in detail later.

[0143] S900 includes:

[0144] S910, take out the standard cell culture multi-well plate 10.

[0145] S920, add 500 μL-1000 μL of culture medium to the standard cell culture multi-well plate 10, continue to culture the cells for a period of time, and observe the delivery effect.

[0146] In one embodiment of the present application, the S700 includes, that is, the driving electrical signal generating unit 200 generates an electrical signal and applies the electrical signal to the electroporation loading unit 100 includes:

[0147] S711, generating a test voltage signal, and transmitting the test voltage signal as an input signal to a test circuit.

[0148] S712, obtaining a feedback voltage signal after the output signal passes through a feedback circuit in the test circuit.

[0149] S713, obtaining a net input signal according to the input signal and the feedback voltage signal.

[0150] S714, determining whether the net input signal meets the accuracy requirement.

[0151] S715, if the net input signal meets the accuracy requirement, the switch assembly 500 is driven to enter a working state to drive the electrical signal generating unit 200 to generate an electrical signal.

[0152] S721 , applying the output signal that meets the accuracy requirement to the electroporation loading unit 100 .

[0153] S722, obtaining the actual voltage of the electroporation loading unit 100, and determining whether the actual voltage of the electroporation loading unit 100 meets the accuracy requirement.

[0154] S723, if the actual voltage of the electroporation loading unit 100 meets the accuracy requirement, then after the preset electroporation time period is continued, the standard cell culture multi-well plate 10 is subsequently taken out.

[0155] S724, if the actual voltage of the electroporation loading unit 100 does not meet the accuracy requirement, the generated voltage signal is corrected, the generated test voltage signal is returned, and the test voltage signal is transmitted to the test circuit as an input signal.

[0156] Specifically, before outputting the electrical signal, this embodiment provides a test logic that first sends out a round of test voltage signals, and determines whether the test voltage signals are normal by comparing the feedback signal with the test voltage signals.

[0157] This embodiment also includes:

[0158] S716, if the net input signal does not meet the accuracy requirement, adjust the waveform and amplitude of the test voltage signal, and return to S711 until the net input signal meets the accuracy requirement.

[0159] One way to determine whether the accuracy requirement is met is to determine whether the voltage effective value of the net input signal is greater than or equal to the first voltage effective value threshold. For example, the first voltage effective value threshold is 970V, which is the threshold we set to meet the accuracy requirement. If the voltage effective value calculation result of the net input signal is 940V, then it is determined that the net input signal does not meet the accuracy requirement and needs to be corrected until the voltage effective value of the net input signal reaches 970V.

[0160] This embodiment further includes S717 executed after S715:

[0161] S717, during the process of outputting the voltage signal, the output voltage signal is compared with the reference voltage in real time, and when an abnormal voltage is found during the comparison, the error value is corrected in real time.

[0162] Specifically, Fig.14 As shown, after the first switch 510 and the second switch 520 are turned on, the voltage signal will enter the high voltage generating unit 220 (i.e. Fig.14 The high voltage module, high voltage module and Figure 1 The high voltage generating unit 220 in the circuit has the same structure as the filtering unit. The high voltage generating unit 220 plays the role of signal amplification, and the filtering unit plays the role of filtering. After the signal is amplified and filtered, it is necessary to compare the amplified and filtered voltage signal with the reference voltage in real time and correct the error.

[0163] There is definitely a certain error gap between the actual voltage of the electroporation loading unit 100 and the ideal voltage we want to apply. One way to determine whether the actual voltage of the electroporation loading unit 100 meets the accuracy requirement is to determine whether the actual voltage of the electroporation loading unit 100 is within a range greater than or equal to the second voltage effective value threshold and less than or equal to the third effective value threshold. For example, the second voltage effective value threshold is 1.1V, and the third voltage effective value threshold is 1.3V. This is the threshold range we set to meet the accuracy requirement. If the detected actual voltage of the electroporation loading unit 100 is 1.15V, which falls within the range of [1.1V, 1.2V], then it is determined that the actual voltage of the electroporation loading unit 100 meets the accuracy requirement.

[0164] For example:

[0165] All circuits are powered on and run, generating a test voltage signal O1. The test voltage signal O1 enters the high voltage generating unit 220 for amplification, and then after filtering, generates an output signal P1 of a large voltage magnitude. Since it is the first time to be powered on and run, the accuracy has not been determined yet, so the switch component 500 (the switch component 500 is Fig.14 The switch in will not be started, so P1 will not pass through the switch component 500, so P1 will not subsequently be divided by two voltage-dividing resistors RF1 and RF2 to generate a voltage signal K1 of a small voltage magnitude, so the net input signal W1=O1-K1=O1-0=O1, and it is determined whether W1 meets the accuracy requirements.

[0166] If W1 does not meet the accuracy requirement, regenerate the test voltage signal.

[0167] If W1 meets the accuracy requirement, then P1 is subsequently divided by two voltage-dividing resistors RF1 and RF2 to generate a voltage signal K1 of a small voltage magnitude.

[0168] After K1 is applied to the electroporation loading part 100, the actual voltage of the electroporation loading part 100 is detected in real time by a voltage detection device, and the actual voltage of the electroporation loading part 100 is compared with the reference voltage to determine whether the accuracy requirement is met.

[0169] If the actual voltage of the electroporation loading unit 100 meets the accuracy requirement, the electroporation operation is performed normally, and after the preset electroporation time period, the standard cell culture multi-well plate 10 is subsequently removed. If the actual voltage of the electroporation loading unit 100 does not meet the accuracy requirement, W1 needs to be corrected, and the test voltage signal O2 is regenerated after the correction.

[0170] The net input signal W2 = O2 - K1, determine whether W2 meets the accuracy requirement, and execute subsequent steps, which will not be repeated here.

[0171] like Fig.14 As shown, Fig.14 The high voltage module in the circuit is the high voltage generating unit 220. The triangle symbol is an operational amplifier, and RF1 and RF2 are two voltage dividing resistors.

[0172] The method of this embodiment can be applied to a test circuit. The test circuit can be set on any electrode plate, and can be set on the first electrode plate 110 or the second electrode plate 120 .

[0173] Xid=X0-XF, Xid is the net input signal, X0 is the input signal, XF is the feedback signal, and V0 is the output voltage signal after being amplified by the high voltage generating unit 220. Fig.14The square structure in the figure is an optocoupler, and the inductor is between the high voltage module and V0. The reference voltage provides a 10V DC voltage.

[0174] In this embodiment, by real-time detection of the voltage waveform during electroporation and correction of errors during electroporation, accurate control of electroporation conditions is achieved, the experimental accuracy of electroporation is improved, and the experimental effect is optimized.

[0175] In one embodiment of the present application, after S700, that is, after driving the electrical signal generating unit 200 to generate an electrical signal and applying the electrical signal to the electroporation loading unit 100, the cell electroporation method further includes:

[0176] S811, obtaining the voltage value output by the early warning circuit.

[0177] S812, determining whether the voltage value output by the early warning circuit is within a preset voltage value range.

[0178] S813: If the voltage value output by the early warning circuit is not within the preset voltage value range, an alarm signal is output.

[0179] Specifically, this embodiment provides an electroporation warning logic. When the standard cell multi-well plate 10 is placed into the electroporation loading part 100, due to the different substances placed in the standard cell multi-well plate 10, the corresponding impedance values ​​are also different, so the introduction of the alarm function helps us detect abnormal situations in advance.

[0180] The voltage value output by the early warning circuit is composed as shown in the following formula 1:

[0181]

[0182] Among them, V OUT The voltage value output by the early warning circuit is the detection value, V i is the voltage value of the porous plate which is an unknown quantity, R2 is the resistance value of R2, R4 is the resistance value of R4, R7 is the resistance value of R7, R8 is the resistance value of R8, R9 is the resistance value of R9, R 10 is the resistance value of R10.

[0183] Therefore, we use voltmeters and other equipment to detect the voltage value output by the early warning circuit. When the voltage value output by the early warning circuit is not within the preset voltage range, it is considered abnormal and an alarm signal is issued.

[0184] Abnormal conditions may be due to improper operation such as idling of the device, incorrect addition of electrochemical material, accidental touch, etc.

[0185] The alarm signal can be a light signal, displayed by lighting, or a sound signal, displayed by setting a buzzer.

[0186] like Fig.13 As shown, Vref is the reference voltage, the value is 1V to 2V, and the preset voltage value range of Vout can be greater than or equal to 1V and less than or equal to 4V. The triangle symbol is not labeled, it is an operational amplifier, the 3.3V part is a power supply that provides a 3.3V DC voltage, and GND is the ground symbol. V porous plate is V i .

[0187] The value of R1 can be 10 ohms, R1 and C1 form an RC filter, and the value of C1 can be 1 nanofarad. The value of R10 can be 100 kilo-ohms ±1%, and the value of C6 can be 10 nanofarad. R10 and C6 form a low-pass filter.

[0188] R7 and C5 form a low-pass filter. The value of R7 can be 82 kilo-ohms ±1%, and the value of C5 can be 10 nanofarads.

[0189] R2 and C3 form a low-pass filter. The value of R2 can be 82 kilo-ohms ±1%, and the value of C3 can be 10 nanofarads.

[0190] The value of R3 may be 1 kilohm, the value of R9 may be 1 kilohm±1%, the value of R5 may be 1 kilohm, the value of C4 may be 10 nanofarads, and the value of R8 may be 10 kilohm±1%.

[0191] In this embodiment, by adding an early warning mechanism, when the entire cell electroporation device is in no-load operation or other improper dangerous operation conditions, the cell electroporation will alarm and stop operating, thereby improving safety and reducing the professional requirements for personnel using the cell electroporation.

[0192] Fig.11 1 is a schematic diagram of assembling a standard cell culture multi-well plate, a first electrode plate 110 , a second electrode plate 120 and a bracket 300 of a cell electroporation device provided in one embodiment of the present application. Fig.11 In the figure, a bottom view of the first electrode plate 110 and a top view of the second electrode plate 120 are also drawn.

[0193] Fig.15 1 is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field to a single cell culture cavity 11 at 100V, 300V, 500V, 800V, 1000V and 1200V respectively.

[0194] Fig.15 The upper left portion (a) is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 100V to a single cell culture cavity 11 . Fig.15(b) in the middle is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 300V to a single cell culture cavity 11. Fig.15 The upper right figure (c) is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 500V to a single cell culture cavity 11.

[0195] Fig.15 The lower left portion (d) is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 800V to a single cell culture cavity 11 . Fig.15 (e) in the lower middle part is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 1000V to a single cell culture cavity 11. Fig.15 The lower right portion (f) is an electric field simulation diagram of the cell electroporation device provided in one embodiment of the present application applying an electric field at 1200V to a single cell culture cavity 11.

[0196] according to Fig.15 It can be seen from the electric field simulation diagram that different colors correspond to different values ​​of electric field strength, and the unit of electric field strength is volt / cm. At 100V, 300V, 500V, 800V, 1000V and 1200V, the color of the electric field formed at the cell culture cavity 11 of the cell electroporation device provided in the present application is uniform, indicating that the electric field is uniform and has a high degree of homogenization.

[0197] Fig.16 1 is a comparison curve of electric field uniformity of a cell electroporation device provided in an embodiment of the present application when an electric field is applied to a single cell culture cavity 11 at 100 V, 300 V, 500 V, 800 V, 1000 V and 1200 V. The uniformity coefficient has no unit.

[0198] according to Fig.15 It can be seen from the electric field simulation diagram that the electric field uniformity fluctuation of the applied electric field at 100V, 300V, 500V, 800V, 1000V and 1200V is in the range of 1.5715 to 1.5716, and the fluctuation is very small, which means that the uniformity of the electric field formed by the cell electroporation device and method provided in the present application is very high.

[0199] Fig.17 The electric field bright field image after applying an electric field to a single cell culture cavity 11 using the cell electroporation device and method provided in one embodiment of the present application and the fluorescence image after electrofection, as well as the electric field bright field image after applying an electric field to a single cell culture cavity 11 using a traditional cell electroporation device and method and the fluorescence image after electrofection.

[0200] Fig.17(a) in the figure has two figures. The figure on the left is a bright field image of the electric field after the cell electroporation device and method provided by the present application applies an electric field to a cell culture cavity 11, and the figure on the right is a fluorescence image after the cell electroporation device and method provided by the present application applies an electric field to a single cell culture cavity 11.

[0201] Fig.17 There are two pictures in (b), the left picture is the electric field bright field picture after the cell electroporation device and method provided by the present application applies an electric field to a cell culture cavity 11, and the right picture is the fluorescence picture after the cell electroporation device and method provided by the present application electrotransfects a single cell culture cavity 11. (a) and (b) are not generated in the same experiment.

[0202] Fig.17 (c) in the figure has two figures. The figure on the left is a bright field image of the electric field after applying an electric field to a cell culture cavity 11 using a traditional cell electroporation device and method, and the figure on the right is a fluorescence image of a single cell culture cavity 11 after electrotransfection using the cell electroporation device and method provided in the present application.

[0203] Fig.17 There are two pictures in (d), the left picture is a bright field picture of the electric field after applying an electric field to a cell culture cavity 11 using a conventional cell electroporation device and method, and the right picture is a fluorescence picture after electrotransfection of a single cell culture cavity 11 using the cell electroporation device and method provided by the present application. (c) and (d) were generated from different experiments.

[0204] Comparing the traditional cell electroporation device and method with the cell electroporation device and method provided in the present application, the number of surviving cells in the electric field bright field image generated by the traditional method is small, while the number of successfully transfected cells in the electric field bright field image generated by the method of the present application is large.

[0205] Comparing the differences between the traditional cell electroporation device and method and the cell electroporation device and method provided in the present application, in the fluorescence image after transfection generated by the traditional method, the number of surviving cells is small, while in the fluorescence image after transfection generated by the method of the present application, the number of successfully transfected cells is large.

[0206] In the fluorescence image after transfection, the bright spots are the cells that have been successfully transfected.

[0207] The technical features of the above-described embodiments may be arbitrarily combined, and the execution order of the method steps is not limited. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0208] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the present application. It should be noted that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A cell electroporation device, characterized in that: For use with standard cell culture multiwell plates, including: An electroporation loading part, used for embedding into a standard cell culture multi-well plate; the bottom surface of the standard cell culture multi-well plate is provided with M cell culture cavities arranged in a matrix, and the cell culture cavities are used for carrying a cell suspension; M is a positive integer and M is greater than or equal to 4; An electrical signal generating unit, used to generate an electrical signal and apply the electrical signal to the electroporation loading unit; The electroporation loading unit comprises: A first electrode plate, the first electrode plate being welded with M first electrode sheets arranged in a matrix; A second electrode plate, the second electrode plate being welded with M second electrode sheets arranged in a matrix; One of the first electrode sheet and the second electrode sheet is a positive electrode sheet, and the other is a negative electrode sheet; The first electrode plate and the second electrode plate are matched and buckled with each other, so that the first electrode plate and the second electrode plate that are closest to each other form a positive and negative electrode group with an internal accommodation space, so that the entire electroporation loading part has M positive and negative electrode groups; When the standard cell culture multi-well plate is embedded in the electroporation loading part, each cell culture cavity is embedded in a receiving space for a positive and negative electrode group aligned with the position of the cell culture cavity, so that the electric signal applied by the positive and negative electrode groups forms an electric field in the receiving space, and the electric field is applied to the cell culture cavity corresponding to the positive and negative electrode groups.

2. The cell electroporation device according to claim 1, characterized in that: The first electrode sheet and the second electrode sheet have the same structure and the same size, the first electrode sheet and the second electrode sheet are symmetrically arranged, and the positive and negative electrode groups composed of the first electrode sheet and the second electrode sheet are surrounded by the cell culture cavity, and the cell culture cavity is a cylindrical shell with a hollow interior; the heights of the first electrode sheet and the second electrode sheet are both greater than the height of the cell culture cavity.

3. The cell electroporation device according to claim 2, characterized in that: The first electrode plate is provided with a plurality of first through holes, the outer diameter of the cell culture cavity is smaller than the diameter of the first through hole, and the outer diameter of the positive and negative electrode groups is smaller than the diameter of the first through hole.

4. The cell electroporation device according to claim 3, characterized in that: Also includes: The bracket is configured as a plate-like structure, and the bracket is provided with M second through holes arranged in a matrix; a first handle in the form of a slat is provided on one side of the bracket, and a second handle in the form of a slat is provided on the other side of the bracket; The M first through holes and the M second through holes are arranged in a one-to-one alignment, that is, each first through hole has a unique second through hole aligned with the first through hole; When the standard cell culture multi-well plate is embedded in the electroporation loading part, the standard cell culture multi-well plate is first interlocked with the bracket to form an assembly, and the assembly is then embedded in the electroporation loading part to complete the embedding of the standard cell culture multi-well plate in the electroporation loading part.

5. The cell electroporation device according to claim 4, characterized in that: The outer diameter of the cell culture cavity is smaller than the diameter of the second through hole.

6. The cell electroporation device according to claim 5, characterized in that: The electroporation loading unit also includes: A plurality of first elastic mechanisms, each of the first electrode sheets is provided with a first elastic mechanism; A plurality of second elastic mechanisms, each second electrode sheet is provided with a second elastic mechanism; A portion of the first elastic mechanism is embedded in the first electrode sheet, and another portion thereof extends out of the outer circumferential surface of the first electrode sheet; A portion of the second elastic mechanism is embedded in the second electrode sheet, and another portion thereof extends out of the outer circumferential surface of the second electrode sheet; The outer diameter of the positive and negative electrode groups is smaller than the diameter of the second through hole; When the assembly is pressed downward, the first elastic mechanism and the second elastic mechanism both move downward into the second through hole, the first elastic mechanism contracts under the action of elasticity and drives the first electrode sheet to move toward the cell culture cavity, the second elastic mechanism contracts under the action of elasticity and drives the second electrode sheet to move toward the cell culture cavity, so that the first electrode sheet and the second electrode sheet clamp the cell culture cavity; the part of the first elastic mechanism extending out of the outer circumferential surface of the first electrode sheet abuts against the inner hole wall of the second through hole, and the part of the second elastic mechanism extending out of the outer circumferential surface of the second electrode sheet abuts against the inner hole wall of the second through hole, so as to maintain the clamping state.

7. The cell electroporation device according to claim 6, characterized in that: Also includes: a switch assembly, electrically connected to the electrical signal generating unit; The switch assembly comprises: A plurality of first switches are arranged on the first electrode plate, each of the first switches is electrically connected to the electrical signal generating unit, and different first drive switches are electrically connected to different columns of first electrode sheets; A plurality of second switches are arranged on the second electrode plate, each of the second switches is electrically connected to the electrical signal generating unit, and different second driving switches are electrically connected to second electrode sheets in different rows.

8. A cell electroporation method, characterized in that: Applied to the cell electroporation device according to any one of claims 1 to 7, the cell electroporation method comprises: A portion of the cell suspension is placed in a cell culture well chamber of a standard cell culture multi-well plate, and a transfection substance is added to the cell culture well chamber; Repeatedly placing a portion of the cell suspension in a cell culture well cavity in a standard cell culture multi-well plate, and adding a transfection substance to the cell culture well cavity, until each cell culture well cavity in a target number of cell culture well cavities has completed the addition of the cell suspension and the transfection substance; Insert a standard cell culture multi-well plate into the electroporation loading section; Set electroporation parameters; Driving the electric signal generating unit to generate an electric signal, and applying the electric signal to the electroporation loading unit; Take out the standard cell culture multiwell plate.

9. The cell electroporation method according to claim 8, characterized in that: The driving electric signal generating unit generates an electric signal and applies the electric signal to the electroporation loading unit, comprising: generating a test voltage signal, and transmitting the test voltage signal as an input signal to a test circuit; After the output signal passes through a feedback circuit in the test circuit, a feedback voltage signal is obtained; A net input signal is obtained according to the input signal and the feedback voltage signal; Determine whether the net input signal meets the accuracy requirements; If the net input signal meets the accuracy requirement, the switch assembly is driven to enter a working state to drive the electrical signal generating unit to generate an output signal that meets the accuracy requirement; Applying an output signal that meets the accuracy requirement to an electroporation loading unit; Obtaining the actual voltage of the electroporation loading unit, and determining whether the actual voltage of the electroporation loading unit meets the accuracy requirement; If the actual voltage of the electroporation loading unit meets the accuracy requirement, the standard cell culture multi-well plate is subsequently removed after the preset electroporation time period is continued; If the actual voltage of the electroporation loading part does not meet the accuracy requirement, the generated voltage signal is corrected, the generated test voltage signal is returned, and the test voltage signal is transmitted to the test circuit as an input signal.

10. The cell electroporation method according to claim 9, characterized in that: After the driving electrical signal generating unit generates an electrical signal and applies the electrical signal to the electroporation loading unit, the cell electroporation method further includes: Obtain the voltage value output by the early warning circuit; Determine whether the voltage value output by the early warning circuit is within a preset voltage value range; If the voltage value output by the early warning circuit is not within the preset voltage value range, an alarm signal is output.

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