CN114214191B - Cell extrusion and electroporation device and electroporation method - Google Patents
Cell extrusion and electroporation device and electroporation method Download PDFInfo
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- CN114214191B CN114214191B CN202111281592.5A CN202111281592A CN114214191B CN 114214191 B CN114214191 B CN 114214191B CN 202111281592 A CN202111281592 A CN 202111281592A CN 114214191 B CN114214191 B CN 114214191B
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- extrusion
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- electroporation
- electroporation device
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- 238000004520 electroporation Methods 0.000 title claims abstract description 83
- 238000001125 extrusion Methods 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000012528 membrane Substances 0.000 claims abstract description 31
- 239000002356 single layer Substances 0.000 claims abstract description 18
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 239000011796 hollow space material Substances 0.000 claims abstract description 10
- 239000010410 layer Substances 0.000 claims description 65
- 238000003825 pressing Methods 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000001890 transfection Methods 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 3
- 239000012737 fresh medium Substances 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 3
- 239000012212 insulator Substances 0.000 claims description 3
- 239000002861 polymer material Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 31
- 239000003364 biologic glue Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005684 electric field Effects 0.000 description 3
- 239000004642 Polyimide Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000005020 polyethylene terephthalate Substances 0.000 description 2
- 229920000139 polyethylene terephthalate Polymers 0.000 description 2
- 229920001721 polyimide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 238000007731 hot pressing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052754 neon Inorganic materials 0.000 description 1
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- -1 polyethylene terephthalate Polymers 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000003466 welding Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
Abstract
The invention discloses a cell extrusion and electroporation device and an electroporation method, wherein the device comprises a consumable with a nano micro-channel substrate and an extrusion electroporation device with a hollow space and a bottom filter membrane, and the extrusion electroporation device is matched with the consumable; the cell-containing culture medium is arranged in the consumable, when the cell-containing culture medium is used, the extrusion electroporation device is in close contact with the consumable, and a single layer is formed between the bottom of the extrusion electroporation device and the bottom of the consumable. According to the cell extrusion and electroporation device and the electroporation method, cells suspended in a culture medium rapidly form a monolayer of cells between electrodes by utilizing a filtering and extrusion mode, so that efficient electroporation is realized.
Description
Technical Field
The invention relates to the field of biological devices, in particular to a cell extrusion and electroporation device and an electroporation method.
Background
Electroporation is a technique in which an exogenous substance is made to enter a cell by breakdown of a cell membrane by an applied electric field. The method has wide application in the fields of gene editing, basic research, protein production, cell therapy and the like. A common electroporation technique is to disperse cells in a buffer solution, place them between cathode and anode electrodes, and then energize them. Because the position of the cells in the electric field under the microcosmic condition and the microenvironment are greatly different, the electroporation process has strong randomness, so that the positive rate is low and the death rate is high. Although the latest commercial electrotometer has made some improvements, for example, the Neon system of Thermo Fisher changes the traditional cuvette electrocuvette cup into an electrocuvette suction head, and the electric field is more uniform by reducing the electrode spacing; in another example, the Nucleofector of Lonza increases the probability of plasmid entry into the nucleus by a special buffer solution, but none of them fundamentally solve the problem of electroporation randomness.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to solve the problems of electroporation randomness of the conventional electroporation apparatus or electroporation apparatus, such as long time consumption, need of using special bio-gel to temporarily adhere cells to a substrate, complex operation, and high cost of bio-gel. Therefore, the present invention provides a cell extrusion and electroporation apparatus and an electroporation method, which rapidly form a monolayer of cells between electrodes by using a filtration and extrusion method, thereby achieving efficient electroporation.
In order to achieve the above object, the present invention provides a cell extrusion and electroporation device, comprising a consumable having a nano-microchannel substrate and an extrusion electroporation device having a hollow space and a bottom filter membrane, wherein the extrusion electroporation device is used in cooperation with the consumable; the cell-containing culture medium is arranged in the consumable, when the cell-containing culture medium is used, the extrusion electroporation device is in close contact with the consumable, and a single layer is formed between the bottom of the extrusion electroporation device and the bottom of the consumable.
Further, the extrusion electroporation device comprises a shell, a hollow space, a filter membrane layer, an electrode layer, a conductive layer, a lower rubber ring, an upper rubber ring and a through hole; the shape of casing is the same and is less than the size of consumptive material with the shape of consumptive material, and the bottom of casing sets up to filter membrane layer and electrode layer, and the conducting layer is inside to be arranged along the lateral wall of casing and top inside, and conducting layer and filter membrane layer, electrode layer enclose into a cavity space, and the lateral wall of casing is provided with the through-hole, and the corresponding position of conducting layer is provided with the through-hole.
Further, the electrode layer is disposed on the upper layer of the filter membrane layer.
Further, the filter membrane layer is provided as a porous filter membrane layer.
Further, the porous filter membrane layer is an insulator filter membrane made of polymer material and having a porous structure, wherein the pore diameter is 0.1-8 μm and the density is 1x10 4 -4x 10 8 /cm 2 The thickness is 5-50 μm.
Further, the electrode layer is provided in a screen structure.
Further, the number of through holes is 2, the 2 through holes are respectively formed in the side wall of the shell and the side wall of the conductive layer of the side wall of the shell and are close to the conductive layer at the top end of the shell, and the 2 through holes are mutually symmetrical.
Further, the device also comprises a sample rack, wherein after the bottom of the extrusion electroporation device and the bottom of the consumable are extruded to form a single layer, the single layer is placed on the sample rack for completing electroporation.
Further, the sample frame includes support body, bottom electrode and connecting electrode circuit, and the bottom electrode setting is provided with a recess in the lower extreme of support body, the upside of bottom electrode, and the recess is used for placing to be changeed thing aqueous solution, and after extrusion electroporation device's bottom and the bottom extrusion of consumptive material formed a individual layer after placing in the sample frame, extrusion electroporation device's bottom and the contact of to be changeed thing aqueous solution in the recess, connecting electrode circuit is connected with conducting layer and bottom electrode respectively, forms the current path.
In another preferred embodiment of the present invention, there is provided a method of cell extrusion and electroporation apparatus comprising the steps of:
1. harvesting a proper amount of cells, dispersing and re-suspending the cells in 50-100 mu L of fresh culture medium to form a cell suspension;
2. adding the cell suspension to the consumable;
3. inserting an extrusion electroporation device at the upper end of the consumable, and slowly pressing until the bottom of the extrusion electroporation device and the substrate of the consumable form a single layer;
4. placing the lower electrode into a frame body of a sample frame, and dripping a proper amount of water solution of a to-be-transferred object into a groove of the lower electrode;
5. placing the extrusion electroporation device and the consumable material in the step 3 into a sample frame, combining with a lower electrode, connecting electrode lines to form a passage, and then completing electroporation by an instrument;
6. the piston-type upper electrode was carefully withdrawn, 50-100. Mu.L of fresh medium was supplied to the consumable, and after a period of continued cell culture, the transfection results were observed.
Technical effects
According to the cell extrusion and electroporation device and method, the preparation time of an electroporation sample is greatly shortened, the use of biological glue is not needed, the damage of the biological glue to cells is avoided, meanwhile, the consumable substrate can be used repeatedly, and the cost is greatly reduced.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic view of a consumable of a cell extrusion and electroporation apparatus according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram showing the extrusion electroporation apparatus of a cell extrusion and electroporation apparatus according to a preferred embodiment of the present invention in combination with a consumable;
FIG. 3 is a schematic diagram of a cell extrusion and electroporation apparatus according to a preferred embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the following description, for purposes of explanation and not limitation, specific details are set forth, such as particular internal procedures, techniques, etc. in order to provide a thorough understanding of embodiments of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present invention with unnecessary detail.
The embodiment of the invention provides a cell extrusion and electroporation device, which comprises a consumable with a nano micro-channel substrate and an extrusion and electroporation device with a hollow space and a bottom filter membrane, wherein the extrusion and electroporation device is matched with the consumable; as shown in FIG. 1, a culture medium 2 containing cells 3 is arranged in a consumable 1, the bottom of the consumable is a substrate with nano micro-channels, in the embodiment, the aperture of the nano micro-channels is 50nm-8 μm, the thickness is 5-10 μm, and the density is 1x10 4 -1x10 8 Individual/cm 2 . When in use, the extrusion electroporation device is tightly contacted with the consumable, and the bottom of the extrusion electroporation device and the bottom of the consumable form a single layer, so thatThe bottom of which is guaranteed to form a single layer by: 1. controlling the amount of injected cells, reducing the likelihood of stacking; 2. the control pitch is small enough that the membrane pitch can only accommodate the height of one cell by extrusion.
As shown in fig. 2, the extrusion electroporation device comprises a housing 5, a hollow space 6, a filter membrane layer 7, an electrode layer 8, a conductive layer 9, a lower rubber ring 10, an upper rubber ring 11 and a through hole 12; as shown in fig. 2, the shape of the housing 5 is the same as the shape of the consumable 1 and smaller than the size of the consumable, and the bottom of the housing 5 is provided with a filter membrane layer 7 and an electrode layer 8, in this embodiment, the electrode layer 8 is provided on the upper layer of the filter membrane layer 7. The filter membrane layer is a porous filter membrane layer. The porous filter membrane layer is an insulator filter membrane with porous structure made of polymer material such as Polycarbonate (PC), polyethylene terephthalate (PET), polyimide (PI), etc., wherein the pore size is 0.1-8 μm, and the density is 1x10 4 -4x 10 8 /cm 2 The thickness is 5-50 μm. The electrode layer is of a screen structure, and is made of one or more of conductive plastic, metal, graphite, etc., with a thickness of 0.1-2mm, a pore diameter of 0.05-0.5mm, and a density of 1x10 2 -2x 10 4 /cm 2 . The mesh electrode layer is in communication with a portion of the conductive layer by way of a connection or wire with the conductive coating. The conducting layer is arranged along the inside of the side wall and the inside of the top of the shell, the conducting layer, the filter membrane layer and the electrode layer enclose a hollow space, the side wall of the shell is provided with a through hole, and the corresponding position of the conducting layer is provided with a through hole. As shown in fig. 2, the number of through holes is 2, 2 through holes are respectively provided on the side walls of the case side wall and the conductive layer of the case side wall and near the conductive layer of the case top end, and 2 through holes are symmetrical to each other. The through holes are used for exhausting air and compressing air.
As shown in fig. 3, the device further comprises a sample holder, wherein after the bottom of the extrusion electroporation device and the bottom of the consumable are extruded to form a single layer, the single layer is placed on the sample holder for completing electroporation.
The sample frame includes support body 13, bottom electrode 14 and connecting electrode circuit 16, 17, and bottom electrode 14 sets up the lower extreme at the support body, and the upside of bottom electrode is provided with a recess, and the recess is used for placing to be changeed thing aqueous solution 15, and after extrusion electroporation device's bottom and the bottom extrusion of consumptive material formed a individual layer placed in the sample frame after, extrusion electroporation device's bottom contacted with to be changeed thing aqueous solution in the recess, connecting electrode circuit is connected with conducting layer and bottom electrode respectively, forms the current path.
In another preferred embodiment of the present invention, there is provided a method of cell extrusion and electroporation apparatus comprising the steps of:
1. harvesting a proper amount of cells, dispersing and re-suspending the cells in 50-100 mu L of fresh culture medium to form a cell suspension;
2. adding the cell suspension to the consumable;
3. inserting an extrusion electroporation device (also called a piston type upper electrode, and manufacturing all components of the extrusion electroporation device into a whole by coinjection, hot pressing, laser welding and other methods) into the consumable, and slowly pressing until the bottom of the extrusion electroporation device and the substrate of the consumable form a single layer;
4. placing the lower electrode into a frame body of a sample frame, and dripping a proper amount of water solution of a to-be-transferred object into a groove of the lower electrode;
5. placing the extrusion electroporation device and the consumable material in the step 3 into a sample frame, combining with a lower electrode, connecting electrode lines to form a passage, and then completing electroporation by an instrument;
6. the piston-type upper electrode was carefully withdrawn, 50-100. Mu.L of fresh medium was supplied to the consumable, and after a period of continued cell culture, the transfection results were observed.
Step 3, inserting a piston type upper electrode at the upper end of the extrusion electroporation device, and slowly pressing until the bottom of the extrusion electroporation device and the consumable substrate form a single layer, wherein the method specifically comprises the following steps:
(1) The pressing down can be accomplished by a machine automatic or manual mode;
(2) In the pressing process, the cell culture medium enters the hollow space part in the electrode through the filter membrane on the bottom surface of the electrode, so that the cells are continuously concentrated;
(3) The rubber ring at the lower end close to the bottom of the electrode plays a role in sealing, so that cell suspension is prevented from overflowing from the side wall;
(4) In the initial stage of pressing, air in the electrode can escape from the through hole under the pushing of the culture medium;
(5) The upper end rubber ring near the upper end of the electrode is sealed in the electrode in the later pressing stage, and the pressure generated by the compressed air enables the filter membrane at the bottom of the electrode to be closer to the cell layer;
(6) After the completion of the pressing, the cells form a monolayer between the consumable substrate and the electrode filter.
In addition, in the step 6, when the transfection result is observed, the cell suspension in the consumable material can be sucked out and transferred to other culture dishes/bottles or multi-well plates for continuous culture. After a period of time, the transfection results were observed.
The cell extrusion and electroporation device and the electroporation method adopt unique designs, and comprise the use of a sealing ring, the use of a through hole air guide, the use of a filter screen and the combination of a screen electrode, so that a cell monolayer is rapidly formed, the randomness of electroporation is greatly reduced, and meanwhile, the preparation time of an electroporation sample is shortened from 8-24 hours to within 15 minutes; the use of biological glue is avoided, the damage to cells is smaller, and the cost is lower; the consumable substrate can be repeatedly used, so that the experiment cost is further reduced.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (2)
1. A cell extrusion and electroporation device, comprising a consumable with a nano-microchannel substrate and an extrusion electroporation device with a hollow space and a bottom filter membrane, wherein the extrusion electroporation device is matched with the consumable; the consumable is internally provided with a culture medium containing cells, and is usedWhen the extrusion electroporation device is in close contact with the consumable, the bottom of the extrusion electroporation device and the bottom of the consumable form a single layer; the extrusion electroporation device comprises a shell, a hollow space, a filter membrane layer, an electrode layer, a conductive layer, a lower rubber ring, an upper rubber ring and a through hole; the shape of the shell is the same as that of the consumable and is smaller than the size of the consumable, the bottom of the shell is provided with the filter membrane layer and the electrode layer, the conducting layer is arranged along the inner part and the inner part of the top of the side wall of the shell, the conducting layer, the filter membrane layer and the electrode layer enclose a hollow space, the side wall of the shell is provided with a through hole, and the corresponding position of the conducting layer is provided with the through hole; the electrode layer is arranged on the upper layer of the filter membrane layer; the filter membrane layer is a porous filter membrane layer; the porous filter membrane layer is an insulator filter membrane with porous structure made of polymer material, wherein the pore diameter is 0.1-8 μm, and the density is 1x10 4 -4x 10 8 /cm 2 The thickness is 5-50 μm; the electrode layer is arranged in a screen structure; the number of the through holes is 2, the 2 through holes are respectively arranged on the side wall of the shell and the side wall of the conductive layer of the side wall of the shell and are close to the conductive layer at the top end of the shell, and the 2 through holes are mutually symmetrical; the bottom of the extrusion electroporation device and the bottom of the consumable material are extruded to form a single layer and then are placed in the sample rack for completing electroporation; the sample frame comprises a frame body, a lower electrode and a connecting electrode circuit, wherein the lower electrode is arranged at the lower end of the frame body, a groove is formed in the upper side of the lower electrode and used for containing an aqueous solution to be transferred, when the bottom of the extrusion electroporation device and the bottom of the consumable are extruded to form a single layer and then placed in the sample frame, the bottom of the extrusion electroporation device is contacted with the aqueous solution to be transferred in the groove, and the connecting electrode circuit is respectively connected with the conducting layer and the lower electrode to form a current path.
2. A method of using the cell extrusion and electroporation device of claim 1, comprising the steps of:
1. harvesting a proper amount of cells, dispersing and re-suspending the cells in 50-100 mu L of fresh culture medium to form a cell suspension;
2. adding the cell suspension to the consumable;
3. inserting the extrusion electroporation device into the upper end of the consumable, and slowly pressing down until the bottom of the extrusion electroporation device and the substrate of the consumable form a single layer;
4. placing a lower electrode into a frame body of a sample frame, and dripping a proper amount of water solution of an object to be transferred into a groove of the lower electrode;
5. placing the extrusion electroporation device and the consumable in the step 3 into a sample holder, combining with a lower electrode, connecting electrode lines to form a passage, and then completing electroporation by an instrument;
6. the piston-type upper electrode was carefully withdrawn, 50-100. Mu.L of fresh medium was supplied to the consumable, and after a period of continued cell culture, the transfection results were observed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111281592.5A CN114214191B (en) | 2021-11-01 | 2021-11-01 | Cell extrusion and electroporation device and electroporation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111281592.5A CN114214191B (en) | 2021-11-01 | 2021-11-01 | Cell extrusion and electroporation device and electroporation method |
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| Publication Number | Publication Date |
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| CN114214191A CN114214191A (en) | 2022-03-22 |
| CN114214191B true CN114214191B (en) | 2024-01-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143436A (en) * | 2013-02-20 | 2015-12-09 | 陈剑 | Methods and devices for electroporation |
| CN106591118A (en) * | 2017-01-05 | 2017-04-26 | 博奥生物集团有限公司 | Cell in-situ electroporation device and use method |
| CN112226365A (en) * | 2020-10-13 | 2021-01-15 | 北京航空航天大学 | Nano-electroporation device based on single cell array and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101598847B1 (en) * | 2014-01-23 | 2016-03-02 | 부경대학교 산학협력단 | Device for micro droplet electroporation via direct charging and electrophoresis, apparatus therefor and method therefor |
| WO2018064463A1 (en) * | 2016-09-30 | 2018-04-05 | University Of Florida Research Foundation, Inc. | Systems and methods including porous membrane for low-voltage continuous cell electroporation |
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2021
- 2021-11-01 CN CN202111281592.5A patent/CN114214191B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143436A (en) * | 2013-02-20 | 2015-12-09 | 陈剑 | Methods and devices for electroporation |
| CN106591118A (en) * | 2017-01-05 | 2017-04-26 | 博奥生物集团有限公司 | Cell in-situ electroporation device and use method |
| CN112226365A (en) * | 2020-10-13 | 2021-01-15 | 北京航空航天大学 | Nano-electroporation device based on single cell array and application thereof |
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