CN114214191A - Cell extrusion and electroporation device and electroporation method - Google Patents
Cell extrusion and electroporation device and electroporation method Download PDFInfo
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- CN114214191A CN114214191A CN202111281592.5A CN202111281592A CN114214191A CN 114214191 A CN114214191 A CN 114214191A CN 202111281592 A CN202111281592 A CN 202111281592A CN 114214191 A CN114214191 A CN 114214191A
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- electroporation
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- 238000004520 electroporation Methods 0.000 title claims abstract description 87
- 238000001125 extrusion Methods 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000012528 membrane Substances 0.000 claims abstract description 28
- 239000000463 material Substances 0.000 claims abstract description 20
- 239000002356 single layer Substances 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 239000011796 hollow space material Substances 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 239000010410 layer Substances 0.000 claims description 63
- 238000003825 pressing Methods 0.000 claims description 16
- 239000006285 cell suspension Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 238000001890 transfection Methods 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 4
- 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
- 230000001502 supplementing effect Effects 0.000 claims 1
- 238000001914 filtration Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 33
- 230000000694 effects Effects 0.000 description 4
- 239000003364 biologic glue Substances 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
- 238000005516 engineering process 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
- 239000013589 supplement Substances 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
- 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
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000005611 electricity Effects 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
- 238000001746 injection moulding 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
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Classifications
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- 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 material with a nanometer 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 material; be provided with the culture medium that contains the cell in the consumptive material, during the use, extrusion electroporation device and consumptive material in close contact with, and the bottom of extrusion electroporation device and the bottom of consumptive material form a monolayer. According to the cell extrusion and electroporation device and the electroporation method, the cells suspended in the culture medium are quickly formed into a monolayer between the electrodes in a filtration 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
The electroporation technology is a technology that the exogenous substances have the opportunity to enter cells by puncturing cell membranes through an external electric field. Has wide application in the fields of gene editing, basic research, protein production, cell treatment and the like. A common electroporation technique is to disperse cells in a buffer solution, place them between a cathode and an anode electrode, and then apply electricity. Due to the fact that the position of the cells in the electric field and the micro-environment are greatly different in a microscopic mode, the electroporation process has strong randomness, and therefore the positive rate is low, and the death rate is high. Although some improvements have been made with the latest commercial electro-rotors, for example, the Neon system of Thermo Fisher changes the traditional cuvette electro-rotor to an electro-rotor tip, making the electric field more uniform by reducing the electrode spacing; in addition, like Lonza's Nucleofector, the probability of plasmid entering the nucleus is increased by a specially prepared buffer solution, but the problem of the randomness of electroporation is not fundamentally solved.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is the randomness of electroporation in the conventional electrotransformation apparatus or electroporation device, but the electroporation device using a single layer of cells for directional electroporation and introduction has problems of long time consumption, temporary adhesion of cells to a substrate using a special bio-gel, complicated operation, high cost of bio-gel, etc. Therefore, the invention provides a cell extruding and electroporating device and an electroporation method, which utilize a filtering and extruding mode to enable cells suspended in a culture medium to quickly form a monolayer of cells between electrodes, thereby realizing efficient electroporation.
In order to achieve the above objects, the present invention provides a cell extrusion and electroporation apparatus, comprising a consumable having a nano microchannel substrate and an extrusion electroporation apparatus having a hollow space and a bottom filter membrane, the extrusion electroporation apparatus being used in cooperation with the consumable; be provided with the culture medium that contains the cell in the consumptive material, during the use, extrusion electroporation device and consumptive material in close contact with, and the bottom of extrusion electroporation device and the bottom of consumptive material form a monolayer.
Further, the extrusion electroporation device comprises a shell, a hollow space, a filter membrane layer, an electrode layer, a conductive layer, a lower end rubber ring, an upper end rubber ring and a through hole; the shape of casing is the same with the shape of consumptive material and is less than the size of consumptive material, and the bottom of casing sets up to filter membrane layer and electrode layer, and the conducting layer is inside along the lateral wall of casing and top inside arrangement, and conducting layer and filter membrane layer, electrode layer enclose into a hollow 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 an upper layer of the filter membrane layer.
Further, the filter membrane layer is set to be a porous filter membrane layer.
Further, the porous filter membrane layer is provided as an insulator filter membrane made of a polymer material and having a porous structure, wherein the pore diameter is 0.1-8 μm, and the density is 1x104-4x 108/cm2The thickness is 5-50 μm.
Further, the electrode layer is provided as a mesh structure.
Further, the through-hole sets up to 2, and 2 through-holes set up the conducting layer that is close to the casing top at the lateral wall of casing lateral wall and the conducting layer of casing lateral wall's lateral wall respectively, 2 through-holes symmetry each other.
Furthermore, the device also comprises a sample frame, and the bottom of the extrusion electroporation device and the bottom of the consumable are extruded to form a single layer and then are placed on the sample frame to complete electroporation.
Further, the sample frame includes the support body, bottom electrode and connecting electrode circuit, and the bottom electrode setting is at the lower extreme of support body, and the upside of bottom electrode is provided with a recess, and the recess is used for placing treats commentaries on classics aqueous solution, places behind the sample frame after the bottom that extrudees the electroporation device and the bottom extrusion of consumptive material form a monolayer, and the bottom that extrudees the electroporation device and wait to change the aqueous solution contact in the recess, and connecting electrode circuit is connected with conducting layer and bottom electrode respectively, forms the current route.
In another preferred embodiment of the present invention, there is provided a method of cell pressing and electroporation apparatus, comprising the steps of:
1. harvesting a proper amount of cells, dispersing and resuspending in 50-100 μ L of fresh medium to form a cell suspension;
2. adding the cell suspension to a consumable;
3. inserting an extrusion electroporation device at 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. putting the lower electrode into the frame body of the sample frame, and dripping a proper amount of water solution to be transferred into the groove of the lower electrode;
5. placing the extrusion electroporation device and the consumable in the step 3 into a sample rack, combining the sample rack with a lower electrode, connecting an electrode circuit to form a passage, and then completing electroporation through an instrument;
6. carefully draw out the piston type upper electrode, supplement 50-100 μ L fresh culture medium to the consumptive material, after continuing to culture the cells for a period of time, observe the transfection result.
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 repeatedly used, and the cost is greatly reduced.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of a consumable of a cell compression and electroporation apparatus according to a preferred embodiment of the invention;
FIG. 2 is a schematic diagram of a cell pressing and electroporation apparatus according to a preferred embodiment of the present invention;
FIG. 3 is a schematic view of a cell pressing and electroporation apparatus according to a preferred embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present 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 merely illustrative of the invention and are not intended to limit 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 the embodiments of the 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 nanometer micro-channel 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; as shown in FIG. 1, a culture medium 2 containing cells 3 is disposed in a consumable 1, the bottom of the consumable is a substrate having a nano-micro channel with a pore size of 50nm-8 μm, a thickness of 5-10 μm, and a density of 1 × 104-1x108Per cm2. During the use, extrusion electroporation device and consumptive material in close contact with, and the bottom of extrusion electroporation device and the bottom of consumptive material form a monolayer, and its bottom is guaranteed to form a monolayer in the following mode of accessible: 1. the injected cell amount is controlled, and the possibility of stacking is reduced; 2. the control spacing is small enough that the membrane spacing can accommodate only one cell height by squeezing.
As shown in fig. 2, the extrusion electroporation apparatus includes a housing 5, a hollow space 6, a filter layer 7, an electrode layer 8, a conductive layer 9, a lower end rubber ring 10, an upper end rubber ring 11, and a through hole 12; as shown in fig. 2, the housing 5 has the same shape as the consumable 1 and is smaller than the consumable, the bottom of the housing 5 is provided with a filter membrane layer 7 and an electrode layer 8, and the electrode layer 8 is disposed on the upper layer of the filter membrane layer 7 in this embodiment. The filter membrane layer is set as a porous filter membrane layer. The porous filter membrane layer is provided as an insulator filter membrane having a porous structure made of a polymer material such as Polycarbonate (PC), polyethylene terephthalate (PET), Polyimide (PI), etc., wherein the pore diameter is 0.1 to 8 μm and the density is 1x104-4x 108/cm2The thickness is 5-50 μm. The electrode layer is of a screen mesh structure, is made of one or more of conductive plastics, metal, graphite and the like, has a thickness of 0.1-2mm, an aperture of 0.05-0.5mm and a density of 1x102-2x 104/cm2. Screen electrode layer passing through andthe conductive coating is connected or wired in communication with portions of the conductive layer. The conducting layer is arranged along the inner part of the side wall and the inner part 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 the through hole. As shown in fig. 2, the number of the through holes is 2, the 2 through holes are respectively arranged on the side wall of the shell and the conductive layer on the side wall of the shell and close to the conductive layer on the top end of the shell, and the 2 through holes are symmetrical to each other. The through holes are used for exhausting air and compressed air.
As shown in FIG. 3, the device further comprises a sample holder, and the bottom of the electroporation device and the bottom of the consumable are extruded to form a single layer, and then the single layer is placed on the sample holder to complete electroporation.
The sample frame includes support body 13, bottom electrode 14 and connecting electrode circuit 16, 17, bottom electrode 14 sets up the lower extreme at the support body, the upside of bottom electrode is provided with a recess, the recess is used for placing treats commentaries on classics aqueous solution 15, place behind the sample frame after the bottom of extrusion electroporation device and the bottom extrusion of consumptive material form a monolayer, the bottom of extrusion electroporation device and the treating commentaries on classics aqueous solution contact in the recess, connecting electrode circuit is connected with conducting layer and bottom electrode respectively, form the electric current route.
In another preferred embodiment of the present invention, there is provided a method of cell pressing and electroporation apparatus, comprising the steps of:
1. harvesting a proper amount of cells, dispersing and resuspending in 50-100 μ L of fresh medium to form a cell suspension;
2. adding the cell suspension to a consumable;
3. inserting an extrusion electroporation device (also called as a piston type upper electrode, and making all parts of the extrusion electroporation device into a whole by methods such as co-injection molding, hot pressing, laser welding and the like) 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. putting the lower electrode into the frame body of the sample frame, and dripping a proper amount of water solution to be transferred into the groove of the lower electrode;
5. placing the extrusion electroporation device and the consumable in the step 3 into a sample rack, combining the sample rack with a lower electrode, connecting an electrode circuit to form a passage, and then completing electroporation through an instrument;
6. carefully draw out the piston type upper electrode, supplement 50-100 μ L fresh culture medium to the consumptive material, after continuing to culture the cells for a period of time, observe the transfection result.
Wherein, step 3, insert piston upper electrode at extrusion electroporation device's upper end, slowly push down, until extrusion electroporation device's bottom and the base of consumptive material form a monolayer, specifically include:
(1) the pressing down can be completed in an automatic or manual mode through a machine;
(2) in the process of pressing down, the cell culture medium enters the hollow space part in the electrode through the filter membrane on the bottom surface of the electrode, thereby continuously concentrating cells;
(3) the lower end rubber ring close to the bottom of the electrode plays a role in sealing, so that the cell suspension is prevented from overflowing from the side wall;
(4) in the initial stage of pressing down, air inside the electrode can escape from the through hole under the pushing of the culture medium;
(5) the upper end rubber ring close to the upper end of the electrode is in the later stage of pressing down, the residual air is sealed in the electrode, 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 depression is completed, the cells form a monolayer between the consumable substrate and the electrode filter.
In addition, in 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. The transfection results were observed over time.
The cell extrusion and electroporation device and the electroporation method provided by the embodiment of the invention adopt unique designs, including the use of a sealing ring, the use of through hole air guide, the use of a filter screen and the combination of a screen electrode, so that a cell monolayer is quickly formed, the randomness of electroporation is greatly reduced, and meanwhile, the preparation time of an electroporation sample is shortened to within 15 minutes from 8-24 hours; avoiding using biological glue, causing less damage to cells and simultaneously having lower cost; the consumable substrate can be used repeatedly, and the experiment cost is further reduced.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A cell extrusion and electroporation device is characterized by comprising a consumable with a nanometer micro-channel 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 cell-containing culture medium is arranged in the consumable, when the consumable is used, the extrusion electroporation device is in close contact with the consumable, and the bottom of the extrusion electroporation device and the bottom of the consumable form a single layer.
2. The cell extrusion and electroporation apparatus of claim 1, wherein the extrusion electroporation apparatus comprises a housing, a hollow space, a filter membrane layer, an electrode layer, a conductive layer, a lower end rubber ring, an upper end rubber ring and a through hole; the shape of casing with the shape of consumptive material is the same and be less than the size of consumptive material, the bottom of casing sets up to filter film layer with the electrode layer, the conducting layer is followed the inside and the inside arrangement in top of the lateral wall of casing, the conducting layer with filter film layer the electrode layer encloses into one hollow space, the lateral wall of casing is provided with the through-hole, the corresponding position of conducting layer is provided with the through-hole.
3. A cell compressing and electroporating device as claimed in claim 2, wherein said electrode layer is disposed on an upper layer of said filter membrane layer.
4. A cell compressing and electroporating device as claimed in claim 2, wherein the filter membrane layer is provided as a porous filter membrane layer.
5. The cell pressing and electroporation apparatus as claimed in claim 4, wherein the porous filter membrane layer is provided as an insulator filter membrane having a porous structure made of a polymer material, wherein the pore size is 0.1 to 8 μm and the density is 1x104-4x108/cm2The thickness is 5-50 μm.
6. A cell pressing and electroporation apparatus as claimed in claim 3, wherein the electrode layer is provided as a mesh structure.
7. A cell compressing and electroporating device as claimed in claim 2 wherein said through holes are provided in 2, 2 through holes are provided in the side wall of the housing and the side wall of said conductive layer of the housing side wall and near said conductive layer of the top end of the housing, respectively, and 2 said through holes are symmetrical to each other.
8. The cell pressing and electroporation apparatus of claim 2, further comprising a sample holder, wherein the bottom of the pressing electroporation apparatus and the bottom of the consumable are pressed to form a monolayer and then placed on the sample holder for electroporation.
9. The cell extrusion and electroporation apparatus according to claim 8, wherein the sample holder includes a holder body, a lower electrode and a connection electrode line, the lower electrode is disposed at a lower end of the holder body, a groove is disposed at an upper side of the lower electrode, the groove is used for placing an aqueous solution to be transferred, when the bottom of the extrusion electroporation apparatus and the bottom of the consumable are extruded to form a single layer and then placed on the sample holder, the bottom of the extrusion electroporation apparatus contacts the aqueous solution to be transferred in the groove, and the connection electrode line is connected to the conductive layer and the lower electrode, respectively, to form a current path.
10. A method of using the cell pressing and electroporation apparatus of any one of claims 1-9, comprising the steps of:
1. harvesting a proper amount of cells, dispersing and resuspending in 50-100 μ L of fresh medium to form a cell suspension;
2. adding the cell suspension to the consumable;
3. inserting the extrusion electroporation device at 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. putting a lower electrode into a frame body of a sample frame, and dripping a proper amount of water solution to be transferred into a groove of the lower electrode;
5. putting the extrusion electroporation device and the consumable in the step 3 into a sample rack, combining the sample rack with a lower electrode, connecting an electrode circuit to form a passage, and then completing electroporation through an instrument;
6. carefully withdrawing the piston-type upper electrode, supplementing 50-100 μ L of fresh culture medium into the consumable, continuing to culture the cells for a period of time, and observing the transfection result.
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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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CN105143436A (en) * | 2013-02-20 | 2015-12-09 | 陈剑 | Methods and devices for electroporation |
US20160333302A1 (en) * | 2014-01-23 | 2016-11-17 | Pukyong National University Industry-University Cooperation Foundation | Electroporation device for transferring material into cells, electroporation apparatus comprising same, and electroporation method |
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 |
US20200032190A1 (en) * | 2016-09-30 | 2020-01-30 | University Of Florida Research Foundation, Inc. | Systems and methods including porous membrane for low-voltage continuous cell electroporation |
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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2021
- 2021-11-01 CN CN202111281592.5A patent/CN114214191B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105143436A (en) * | 2013-02-20 | 2015-12-09 | 陈剑 | Methods and devices for electroporation |
US20160333302A1 (en) * | 2014-01-23 | 2016-11-17 | Pukyong National University Industry-University Cooperation Foundation | Electroporation device for transferring material into cells, electroporation apparatus comprising same, and electroporation method |
US20200032190A1 (en) * | 2016-09-30 | 2020-01-30 | University Of Florida Research Foundation, Inc. | Systems and methods including porous membrane for low-voltage continuous cell 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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