CN116590143A - Experimental consumable for implementing nano electrotransfection on cells in vitro - Google Patents
Experimental consumable for implementing nano electrotransfection on cells in vitro Download PDFInfo
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- CN116590143A CN116590143A CN202310619850.9A CN202310619850A CN116590143A CN 116590143 A CN116590143 A CN 116590143A CN 202310619850 A CN202310619850 A CN 202310619850A CN 116590143 A CN116590143 A CN 116590143A
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- 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
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- C12M23/00—Constructional details, e.g. recesses, hinges
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Abstract
The invention discloses an experimental consumable for implementing nano electrotransfection on cells in vitro, which comprises a top cover, a cell cavity and a base, wherein the top cover is provided with a positive electrode, the cell cavity comprises a non-conductive side wall and a nano-pore chip connected with the bottom of the side wall, and the base is provided with a negative electrode and a delivery cavity. According to the invention, the consumable is arranged into three parts, the cell cavity and the delivery cavity separation structure provide enough space for a user, and the solution can be easily injected into the target position of the cell cavity by the pipette in the state that the three components are disassembled, so that the operations of cell digestion and liquid exchange are convenient.
Description
Technical Field
The invention relates to biological experiment consumable material, which is matched with an instrument to complete the operation of nanometer electrotransfection experiment on cells in vitro.
Background
Nanoelectrotransfection, also known as "nanotunneling electroporation", english Nano Electroporation, NEP for short, is a technique developed by James Lee team, professor James Lee, ohio state university, usa. The application range of the technology is cell therapy, gene editing and therapy, transdermal drug delivery and the like. In the conventional electrotransfection technology, an electric pulse load is applied to the whole cell solution, so that a cell membrane is instantaneously opened and closed under electric stimulation, and target substances carrying negative charges in the solution are delivered into the cell at the moment. In this cell solution, the cells are in a uniformly distributed suspended state; the target substance to be delivered, including DNA, RNA (molecular weight up to the kD level), etc., is also in a homogeneous state; in addition, manufacturers optimize the composition of the electrotransport fluid reagent in order to increase the uniform distribution of the voltage load throughout the solution. The positive electrode and the negative electrode are directly contacted with the cell solution, and the electrode material is a material with better biocompatibility such as aluminum, platinum or conductive polymer; the applied voltage is in the range of 100-1000V. The existing cell electrotransfection products in the market are all based on the traditional electrotransfection technology, and are matched with the instrument and the consumable. The core function of the instrument is electric pulse output; the consumable is a container for bearing a cell solution, and the electrode is integrated on the consumable and is in direct contact with the cell; the instrument is provided with a positive and negative voltage output module which is in direct contact with the electrode on the consumable material so as to realize that the voltage is applied to the cell solution. Traditional electrotransfection consumables come in a variety of forms: 1. non-GMP grade: (a) an electrorotor containing 100-200ul of cell solution; (b) 96-well electrotransfer plates, each well capable of holding 10-20ul of cell solution; or 384 well electrotransfer plates, etc. Gmp grade: electrotransfection chips suitable for large-scale GMP production. The related patent can be referred to the patent application of China patent application No. 202011093674.2, the publication date is 2021, 1 month and 15 days, and a nano electroporation device based on a single cell array is disclosed, which comprises: the three-dimensional micro-nano structure chip is used for arraying single cells and providing nano channels for molecular transmission; the cell culture chamber is positioned above the three-dimensional micro-nano structure chip and is used for cell culture; the biomolecule storage chamber is positioned below the three-dimensional micro-nano structure chip and is used for storing biomolecules; a top electrode above the cell culture chamber for forming a circuit when electroporated; and the bottom electrode is positioned below the biomolecule storage chamber and is used for forming a loop when electroporation is performed. In the scheme, the nano electroporation device is of an integral structure, the cell solution and the delivery solution are inconvenient to pipette, the integral detection fault tolerance is low, and the control is not easy to master.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art, and provide an experimental consumable for implementing nano electrotransfection on cells in vitro, wherein the experimental consumable provides reliable solution operation and high fault tolerance.
The experimental consumable for implementing nano electrotransfection on cells in vitro comprises a top cover, a cell cavity and a base, wherein the top cover is provided with a positive electrode, the cell cavity comprises a non-conductive side wall and a nano-pore chip connected with the bottom of the side wall, and the base is provided with a negative electrode and a delivery cavity.
The experimental consumable for implementing the nano electrotransfection on the cells in vitro provided by the invention also has the following technical characteristics:
further, the top cover is made of conductive material or assembled by nonconductive material and conductive material, and the base is made of conductive material or assembled by nonconductive material and conductive material.
Further included is a positive electrode on the top cover extending toward the bottom of the cell cavity, the positive electrode contacting a cell solution at the bottom of the cell cavity.
The top cover is characterized in that a hollow pipe column body is formed in the middle of the top cover, the positive electrode is arranged in the pipe column body, and the bottom of the positive electrode protrudes out of the pipe column body.
Further comprises a cell solution accommodating cavity formed by inward contraction of the lower part of the side wall of the cell cavity, and the nanopore chip is positioned at the bottom of the cell solution accommodating cavity.
Further comprising, the positive electrode covers the top of the cell solution containing chamber, and a convex ring is formed on the side wall of the cell chamber.
The cell cavity is characterized by further comprising a threaded or rubber ring arranged between the top cover and the cell cavity, and a threaded or rubber ring is arranged between the base and the cell cavity.
Further comprises a cell cavity liquid injection hole which is arranged on the top cover or the cell cavity and communicated with the cell cavity.
Further comprises a delivery cavity liquid injection hole which is arranged on the base or the cell cavity and communicated with the delivery cavity.
The nano-pore chip is etched by a silicon-based material or made of a non-conductive and highly hydrophilic material, and a plurality of nano-pores are formed on the nano-pore chip.
Compared with the prior art, the experimental consumable for implementing the nano electrotransfection on the cells in vitro has the following advantages: according to the invention, the consumable is arranged into three parts, the cell cavity and the delivery cavity separation structure provide enough space for a user, and the solution can be easily injected into the target position of the cell cavity by the pipette in the state that the three components are disassembled, so that the operations of cell digestion and liquid exchange are convenient. In addition, the added liquid injection hole allows the assembly to be filled with liquid by using a liquid-transferring gun or an injection agent under the condition that the cavity is hollow, so that the cavity is easy to be filled and bubbles are conveniently avoided. After the transfection experiment is completed, the cell cavity can be used for directly observing the whole nanopore chip under a microscope, and observing cells, particularly adherent cells. Cells may be kept in the cell cavity for a long period without contact with the conductive material, only during transfection, so that cells may be cultured and passaged in the cell cavity for a long period. The consumable material and nano electrotransfection technology can achieve the transfection effect of multiple cells with high efficiency and high activity.
Drawings
Fig. 1 is a front view of the present invention.
Fig. 2 is a perspective view of the present invention.
Fig. 3 is a perspective view of the top cover of the present invention.
FIG. 4 is a front view of a cell cavity according to the present invention.
FIG. 5 is a perspective view of a cell cavity according to the present invention.
Fig. 6 is a perspective view of a base in the present invention.
Fig. 7 is a cross-sectional view of the present invention.
Fig. 8 is a cross-sectional exploded view of the present invention.
Detailed Description
For clarity of explanation of the aspects of the present invention, preferred embodiments are given below in detail with reference to the accompanying drawings. The following description is merely exemplary in nature and is not intended to limit the application or uses of the present disclosure. It should be understood that throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.
As shown in fig. 1 to 8, the experimental consumable for performing nano-electrotransfection on cells in vitro provided by the invention comprises a top cover 1, a cell cavity 2 and a base 3, wherein the top cover 1 is provided with a positive electrode 4, the cell cavity 2 comprises a non-conductive side wall 21 and a nano-pore chip 5 connected with the bottom of the side wall 21, and the base 3 is provided with a negative electrode 6 and a delivery cavity 31. The negative electrode 6 is connected to the delivery chamber 31, and the delivery substance solution in the delivery chamber 31 is in contact with the negative electrode 6. The invention is designed into three independent detachable parts based on the application scene of biological experiments, the top cover 1 covers the top of the cell cavity 2, and the top of the cell cavity 2 is opened. The lower part of the cell cavity 2 is connected with the base 3, a delivery cavity 31 in the base 3 is in contact with the nanopore chip 5, and a delivery substance solution in the delivery cavity 31 covers the top of the nanopore chip 5. The invention adopts an assembly structure of three parts, provides enough open space for users, and the liquid-transferring gun can easily inject the solution into the target position of the cell cavity in the state of disassembling the three components, thus being convenient for cell digestion and liquid-changing operation. After the transfection experiment is completed, the cell cavity can be used for directly observing the whole nanopore chip under a microscope, and observing cells, particularly adherent cells. Cells may be kept in the cell cavity for a long period without contact with the conductive material, only during transfection, so that cells may be cultured and passaged in the cell cavity for a long period. The consumable material and nano electrotransfection technology can achieve the transfection effect of multiple cells with high efficiency and high activity.
Nano electrotransfection technique: the cells and delivery material are no longer mixed together, but rather are separated on both sides of a nanopore chip; the positive and negative electrodes are in contact with the cell solution and the delivery substance solution; attaching cells to the surface of the nanopore by some cell manipulation techniques; under the stimulation of pulse signals, cell membranes are instantaneously opened, and a delivery substance enters cells through the nanopores and the cell membrane openings; the voltage is below 100V; the transfection efficiency and activity of the cells are improved compared with the traditional electrotransfection technology.
Referring to fig. 1 to 8, in the above embodiment of the present invention, the top cover 1 is made of a conductive material or assembled of a nonconductive material and a conductive material, and the base 3 is made of a conductive material or assembled of a nonconductive material and a conductive material. The conductive material is a more conventional metal material such as copper, aluminum, platinum or conductive polymer. The non-conductive material adopts more conventional plastics and the like, and the conductive material and the non-conductive material in the invention can be arbitrarily selected according to the requirements.
Referring to fig. 1 to 8, in the above embodiment of the present invention, it is further included that the positive electrode 4 on the top cover 1 extends toward the bottom of the cell cavity 2, and the positive electrode 4 contacts the cell solution 10 located at the bottom of the cell cavity 2. The cap 1 of the present invention is required to have a conductive function in addition to the function of keeping the cell solution clean. The positive electrode 4 needs to be in complete contact with the cell solution, so that the uniformity of the conductivity of the cell solution is ensured. I.e. the contact surface of the positive electrode 4 with the cell solution is substantially equal to the liquid level of the cell solution. The positive electrode 4 extrudes the cell solution, so that more cell solution can be extruded, and cells can be spread on the surface of the nanopore chip 5 as much as possible.
Referring to fig. 1 to 8, in the above embodiment of the present invention, further comprising, a hollow column 11 is formed at the middle of the top cap 1, the positive electrode 4 is disposed in the column 11, and the bottom of the positive electrode 4 protrudes from the column 11. The column 11 in this embodiment is a cylinder, and the positive electrode 4 is also a cylinder. When the cap 1 is assembled with the cell cavity 2, the column 11 is inserted into the cell cavity 2. The column 11 facilitates the fixing of the positive electrode 4.
Referring to fig. 1 to 8, in the above-described embodiment of the present invention, it is further included that the lower portion of the sidewall 21 of the cell chamber 2 is contracted inward to form a cell solution accommodating chamber 22, and the nanopore chip 5 is located at the bottom of the cell solution accommodating chamber 22. The positive electrode 4 covers the top of the cell solution containing chamber 22, and a convex ring 23 is formed on the side wall 21 of the cell chamber 2. The positive electrode 4 and the cell solution containing cavity 22 form a standard containing cavity, so that the added cell solution is of standard volume, and accurate reaction volume and solution impedance are maintained. The convex ring 23 is convenient to hold and use. The cell cavity 2 in the invention can be placed in a culture dish or assembled with special upper and lower dust covers and cultured together in a cell incubator. The cell cavity 2 can be used as a separate component without frequent pipetting.
Referring to fig. 1 to 8, in the above embodiment of the present invention, a screw thread or a rubber ring is further provided between the top cover 1 and the cell cavity 2, and a screw thread or a rubber ring is provided between the base 3 and the cell cavity 2. In this embodiment, a rubber ring structure is adopted, a groove 24 is formed on the side wall 21 of the cell cavity 2, and a rubber ring 25 is disposed in the groove 24. The above-mentioned cooperation structure utilizes the frictional force of rubber circle 25 to realize the connection of three, and this kind of connected mode resistance is suitable, if the frictional force of connection is too big, then needs great power to open the three, leads to the fact the influence to the cell solution easily. The friction force is too small, so that the connection of the three components is not stable enough, and the use is affected.
Referring to fig. 1 to 8, in the above embodiment of the present invention, it is further included that a cell cavity filling hole 8 is provided on the top cover 1 or the cell cavity 2, and the cell cavity filling hole 8 is in communication with the cell cavity 2. In this embodiment, the cell cavity liquid injection hole 8 is disposed on the top cover 1. A delivery cavity liquid injection hole 9 is arranged on the base 3 or the cell cavity 2, and the delivery cavity liquid injection hole 9 is communicated with the delivery cavity 31. The liquid injection hole 9 of the delivery cavity in the embodiment is arranged on the base 3. Through the cell cavity liquid injection hole 8, a solution such as DPBS, PBS or cell culture medium is injected into the cell solution accommodating cavity 22 in the cell cavity 2, the cell solution accommodating cavity 22 is filled, and the specific solution is determined by the specific condition of the cell. The delivery substance solution is injected into the delivery chamber 31 through the delivery chamber injection hole 9, filling the delivery chamber 31. The present invention can first assemble the top cover 1, the cell cavity 2 and the base 3 together, and then inject the solution through the cell cavity injection hole 8 and the delivery cavity injection hole 9, thereby filling the solution cavity. The injection of the solution can reduce the generation of bubbles.
Referring to fig. 1 to 8, in the above embodiment of the present invention, the nanopore chip is etched from a silicon-based material or is made from a non-conductive and highly hydrophilic material, and the nanopore chip has a plurality of nanopores thereon. The specific structure and principle of the nanopore chip can be seen in patent application of patent application number 202011093674.2 of China, and details are not repeated here. The nanopore chip 5 is laser bonded to the sidewall 21.
In the experimental operation process, the basic flow of the cell experiment carried out by the consumable is as follows:
1. a cell solution 10 and a delivery substance solution are prepared at a certain specific concentration.
2. A specific volume of cell solution is moved into the cell cavity.
3. The cell cavity carrying the cells can be placed in a petri dish or assembled with a special upper and lower dust cap and cultured together in a cell incubator, which is not necessary.
4. The cell cavity, the base and the top cover are assembled in an exchangeable specific sequence, and the stability of the assembly of the two parts is ensured by the screw threads or the rubber rings and the like between every two parts.
5. Through the cell cavity liquid injection hole, the solution of DPBS, PBS or cell culture medium is injected into the cell cavity to fill the cell cavity, and the specific solution is determined by the specific condition of the cell. The delivery substance solution is injected into the delivery lumen through the delivery lumen injection port, filling the delivery lumen.
6. And placing the assembled consumable in an electrotransfection instrument or connecting the assembled consumable with a consumable adapter externally connected with the instrument, so that the instrument applies electric energy load to the consumable.
In summary, the foregoing is provided merely for illustrating the principles of the present invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An experimental consumable for implementing nano electrotransfection on cells in vitro, which is characterized in that: the cell cavity comprises a non-conductive side wall and a nanopore chip connected with the bottom of the side wall, and the base is provided with a negative electrode and a delivery cavity.
2. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: the top cover is made of conductive materials or assembled by nonconductive materials and conductive materials, and the base is made of conductive materials or assembled by nonconductive materials and conductive materials.
3. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: the positive electrode on the top cover extends towards the bottom of the cell cavity, and the positive electrode contacts the cell solution at the bottom of the cell cavity.
4. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 3, wherein: the middle part of the top cover is formed with a hollow pipe column body, the positive electrode is arranged in the pipe column body, and the bottom of the positive electrode protrudes out of the pipe column body.
5. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 3, wherein: the lower part of the side wall of the cell cavity is contracted inwards to form a cell solution accommodating cavity, and the nanopore chip is positioned at the bottom of the cell solution accommodating cavity.
6. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 5, wherein: the positive electrode covers the top of the cell solution containing cavity, and a convex ring is formed on the side wall of the cell cavity.
7. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: a thread or a rubber ring is arranged between the top cover and the cell cavity, and a thread or a rubber ring is arranged between the base and the cell cavity.
8. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: the top cover or the cell cavity is provided with a cell cavity liquid injection hole, and the cell cavity liquid injection hole is communicated with the cell cavity.
9. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: the base or the cell cavity is provided with a delivery cavity liquid injection hole, and the delivery cavity liquid injection hole is communicated with the delivery cavity.
10. An experimental consumable for the in vitro nanoelectrotransfection of cells according to claim 1, wherein: the nano-pore chip is etched by a silicon-based material or made of a non-conductive and highly hydrophilic material, and is provided with a plurality of nano-pores.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310619850.9A CN116590143A (en) | 2023-05-29 | 2023-05-29 | Experimental consumable for implementing nano electrotransfection on cells in vitro |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310619850.9A CN116590143A (en) | 2023-05-29 | 2023-05-29 | Experimental consumable for implementing nano electrotransfection on cells in vitro |
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| CN116590143A true CN116590143A (en) | 2023-08-15 |
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| CN202310619850.9A Pending CN116590143A (en) | 2023-05-29 | 2023-05-29 | Experimental consumable for implementing nano electrotransfection on cells in vitro |
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- 2023-05-29 CN CN202310619850.9A patent/CN116590143A/en active Pending
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