CN110527624A - A kind of intermittent streaming electrotransfection device - Google Patents
A kind of intermittent streaming electrotransfection device Download PDFInfo
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- CN110527624A CN110527624A CN201810493935.6A CN201810493935A CN110527624A CN 110527624 A CN110527624 A CN 110527624A CN 201810493935 A CN201810493935 A CN 201810493935A CN 110527624 A CN110527624 A CN 110527624A
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- 239000007788 liquid Substances 0.000 claims abstract description 36
- 230000005611 electricity Effects 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 description 36
- 210000000170 cell membrane Anatomy 0.000 description 9
- 239000000126 substance Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
- 238000005424 photoluminescence Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000010408 film Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000002433 hydrophilic molecules Chemical class 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
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- 239000010409 thin film Substances 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
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
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Abstract
The present invention provides a kind of vertical intermittent formula streaming electrotransfection device, including cavity, first electrode, second electrode, the first conduit, the second conduit；The first electrode and second electrode are respectively placed in the symmetrical wall of cavity, if liquid in-out mouth, on side wall, electrode is just in top and bottom, and first electrode and second electrode can be electrically connected by the upper and lower ends of cavity with outside formation；Inlet and liquid outlet are set on the cavity wall, and the inlet and the first conduit are tightly connected, and the liquid outlet and the second conduit are tightly connected.The device of the invention is shocked by electricity using sectional intermittent formula feed liquor, guarantees that every chamber sample obtains more uniform electric shock, before liquid enters, negative pressure of vacuum is formed in cavity, liquid can be full of cavity after entering, reduce the generation of electrotransfection bubble.
Description
Technical field
The present invention relates to electrotransfection fields, are related to a kind of intermittent streaming electrotransfection device.
Background technique
Cell membrane is the thin film for being enclosed in pericellular, be cell with the external world carry out selective substances exchange it is penetrating
Property barrier.Cell membrane makes cell become an independent living unit, and possesses a metastable interior environment.Ambient enviroment
In some substances can be by cell membrane, other substances are then not all right.Cell can be absorbed by cell membrane from ambient enviroment
Nutriment is discharged metabolite, the transhipment of substance is made to reach equilibrium state.So the basic function of cell membrane is exactly to maintain cell
Interior microenvironment relatively stable simultaneously selectively carries out mass exchange with external environment.
The study found that if applying the electro photoluminescence of some strength to cell and continuing for some time, so that it may inducing cell
Some micropores are generated on film, enhance the permeability of cell, and so-called cell electroporation just refers to that cell is adding impulse electric field outside
Under effect, the biophysics process of instantaneous micropore is formed on cell membrane lipid bilayer.
When electroporation occurs for cell membrane, permeability and membrane conductance can instantaneously increase, and make hydrophilic molecules, DNA, albumen
Matter, virion, drug granule etc. cannot get enter into cell by the molecule of cell membrane under normal circumstances.It removes in a short time
After electro photoluminescence, cell membrane can become again selective permeation barrier with self-recovery.Turn with traditional chemical transfection and virus
Dye is compared, since electrotransfection has many advantages, such as no chemical contamination, permanent damage, efficiency will not be caused higher cell, in life
The fields such as object physics, molecular biology, clinical medicine have broad application prospects.
Although the mechanism of electrotransfection effect is not entirely clear that, cell electrotransfection herein is well known, including thin
The movement of after birth lipid bilayer molecule causes to form temporary micropore on film, allows exogenous molecule thin by diffusing into
Born of the same parents.
In the prior art, three classes method mainly completes the process of cell electrotransfection:
Cell is placed in a pair of between several millimeters to several centimeters parallel pole.Make the electricity of cell between the electrodes
By electro photoluminescence in, to realize the purpose of electrotransfection.For example, United States Patent (USP) US5389069.
It is penetrated in tissue or cell using miniature needle electrode and is shocked by electricity to cell, achieve the purpose that electrotransfection.For example,
Chinese patent CN 103865794B.
One chamber is placed between a pair of of parallel pole, while so that the aaerosol solution of cell flows in the chamber
It is shocked by electricity.For example, United States Patent (USP) US6773669.
In disclosed technology, electrotransfection is frequently in carry out in single chamber test tube, is used for the maximum capacity of electrotransfection
Usually 1ml.In the occasion for needing to handle a large amount of samples, this technology tedium, large labor intensity.
Researcher on this basis, has invented the form of multiple electrotransfection chamber parallel connections, and this technology has its advantage, but not
The difficulty for being difficult to realize quickly handle a large amount of samples can be solved at all.
The present invention carries out electrotransfection aiming at the problem that current electrotransfection cavity treating capacity is limited to, using intermittent feed liquor, protects
It demonstrate,proves every chamber sample and obtains more uniform electrotransfection, before liquid enters, form negative pressure of vacuum in cavity, liquid is full of cavity after entering
After carry out electrotransfection, the generation of bubble when reducing electrotransfection, after the completion of electrotransfection, liquid flows out cavity, and cavity is formed again to be born
Pressure repeats intermittent feed liquor.
Summary of the invention
The present invention be directed to above-mentioned application limitations of the existing technology, provide a kind of intermittent streaming electrotransfection dress
It sets.
The device of the invention can and be not limited to liquid full of electrotransfection is carried out after cavity, the shape of streaming can also be formed
State.
In order to achieve the above object, the technical solution of intermittent streaming electrotransfection device provided by the present invention is summarized such as
Under: a kind of vertical intermittent formula streaming electrotransfection device, including cavity, first electrode, second electrode, the first conduit, the second conduit；
It is characterized in that, the first electrode and second electrode are respectively placed in the symmetrical wall of cavity, and first electrode and second electrode
It can be electrically connected by the side wall of cavity with outside formation；Inlet and liquid outlet, the feed liquor are set on the cavity wall
Mouth is tightly connected with the first conduit, and the liquid outlet and the second conduit are tightly connected.
The first electrode is placed in the top of cavity, and the second electrode is placed in the bottom of cavity, and this mode requires liquid
Body just can be carried out electrotransfection after must being full of cavity.
The first electrode is placed in the left side of cavity, and the second electrode is placed in the right side of cavity, and this mode is conducive to
Make being lower in the height of cavity for suspension cell, is affected by gravity smaller
The inlet is set to the bottom of cavity wall, and the liquid outlet is set to the top of cavity wall.Inlet
It is identical with the conduit caliber on liquid outlet.In at the same time, guarantee that liquid inlet and outlet rate is consistent.
One-way control valve is set on first conduit, one-way control valve is set on second conduit.
The containment portion setting makes the device of intracavity liquid mixing, and cell is in flow regime, cell in holding chamber body
It is evenly distributed.
Described device further includes pump, can choose peristaltic pump etc..
Vertical intermittent formula streaming electrotransfection device of the invention, the cavity are prepared in such a way that mold pours, and make institute
State first electrode and second electrode i.e. embeddable cavity during cavity preparation.
Technical solution of the present invention bring the utility model has the advantages that
Intermittent streaming electrotransfection device of the invention is capable of handling a large amount of fluid samples.
Intermittent streaming electrotransfection device of the invention, before liquid feed liquor, cavity forms negative pressure, and liquid can be full of chamber
Body, electrotransfection are able to suppress bubble generation in the process, and electrotransfection efficiency fluctuation caused by reducing because of bubble attachment improves electrotransfection
The stability of device.
Intermittent streaming electrotransfection apparatus structure of the invention is novel in design, has initiative.
Detailed description of the invention
Fig. 1 vertical intermittent formula streaming electrotransfection device exemplary diagram of the present invention；
Fig. 2 electric revolving cup schematic diagram in the prior art；
Appended drawing reference: 1- cavity, 2- first electrode, 3- second electrode, 4- inlet, 5- liquid outlet, the first conduit of 6-, 7-
Second conduit, 8- one-way control valve, 9- one-way control valve, 10- insulating layer, 11- electrode slice, 12- electrode cup lid.
Specific embodiment
Embodiment one
Overall construction design
The vertical intermittent formula streaming electrotransfection device that the present invention designs is as shown in Figure 1, the top and bottom of the cavity are divided
Not She Zhi first electrode 2 and second electrode 3, inlet 4, the top on the side wall of left side is arranged in the top on the left of the cavity on side wall
Liquid outlet 5 is arranged in portion, and the inlet and the first conduit 6 are tightly connected, and can connect and fills sample sample sack, the liquid outlet and the
Two conduits 7 are tightly connected, and be can connect and are received sample sample sack.It is respectively set on first conduit and the second conduit unidirectionally controlled
Valve.
Before liquid enters cavity, the first supravasal one-way control valve 8 is closed, the second supravasal one-way control valve 9
It opens, the intracorporal gas of chamber is discharged using pump, is then shut off the second supravasal one-way control valve 9.It opens on the first conduit
One-way control valve 8, target liq sample enters inside cavity from inlet, after cavity is full of, close the first supravasal list
To control valve 8.
After the completion of electrotransfection, the one-way control valve 9 of the second conduit on liquid outlet is opened, according to gravity factor, liquid meeting
It is flowed into from top inlet 4 to congregate, thus target liq sample can be discharged from bottom liquid outlet 5, target liq sample
After being completely exhausted out, the one-way control valve 9 of the second conduit is closed, and the first supravasal one-way control valve 8 is opened, and more than repetition
Movement.
Embodiment two
Liquid batch formula feed liquor mode
It opens one-way control valve 9 and gas in cavity is discharged to clean, closing one-way control valve 9；Open one-way control valve 8 into
Fluid valve is entered target liq sample inside cavity 1 from inlet 4, using peristaltic pump according to the flow velocity of 5ml/min to mesh
After standard liquid body is full of cavity, one-way control valve 8 is closed, forms airtight cavity, carries out electrotransfection.It is unidirectional to control after the completion of electrotransfection
Valve 9 processed is opened, and continues to be extracted target liq sample out from liquid outlet 5 according to 5ml/min with peristaltic pump, and being sufficiently will be after electrotransfection
Target liq sample be cleaned, one-way control valve 8 can be opened and increase additional liquid inlet volume and develop target liq sample,
Last one-way control valve 9 is closed, and one-way control valve 8 is opened, and operation more than repetition.
Embodiment three
Biological test experiment
Collect the CHO-S cell (Chinese hamster ovary cell, floating type) for being in logarithmic growth phase, revolving speed 1000rpm/
Min is centrifuged 5min, abandons supernatant, and electricity consumption turns buffer and cell is resuspended, so that the density of cell is 1 × 107A/ml is added and needs
Electrotransfection is transferred to the plasmid pCDNA3.1-GFP of cell, makes the 20 μ g/ml of concentration of plasmid, soft to be uniformly mixed.
Cooperate electrotransfection instrument use, using following condition carry out electro photoluminescence: flow velocity 5.5ml/min, 240 volts of voltage,
6 milliseconds of pulse width, pulse number 2 times, the pulse spacing 1 second carry out electricity and turn.After electrotransfection, the cell after transfection is hanged
Liquid is placed in centrifuge tube, 1000rpm/min, is centrifuged 5min.Supernatant is abandoned, OptiCHO culture medium is added, cell, inoculation and three is resuspended
Pyramid culture in glassware, culture density 2 × 106/ ml is subsequently placed on shaking table and cultivates, shaking speed 125rpm/min, cultivates item
Part: 37 DEG C of temperature, gas concentration lwevel 8%.In fluorescence microscopy microscopic observation transfection efficiency after 24 hours.
Respectively using the vertical intermittent formula streaming electricity rotary device of the invention being prepared and the prior art known in the art
In electrotransfection cup (shown in Fig. 2) carry out biological test experiment as comparison device, each device be arranged 6 it is parallel repeat, compare
Electrotransfection efficiency and cell survival rate, electrotransfection efficiency pass through BD flow cytomery；Cell survival rate is contaminated by trypan blue
Color detection.
The suspension of cell, plasmid that vertical intermittent formula streaming electricity rotary device of the invention will be mixed by peristaltic pump passes through
Inlet 4 injects cavity, then carries out electrotransfection, after the completion of electrotransfection, is extracted out by liquid outlet 5.
The suspension of the cell mixed, plasmid is moved into electrotransfection cup in the prior art by pipette, covers cup
It covers, progress electrotransfection, after the completion of electrotransfection, suspension is removed, electrotransfection efficiency and cell survival rate are detected.
Culture observation carried out to electrotransfection cell, the average electrotransfection efficiency of electricity revolving cup in the prior art 78.3%,
Mean percent cell survival is 89.1%；88.6%, mean percent cell survival exists the average electrotransfection efficiency of apparatus of the present invention
98.1%.
Experimental result is as follows:
| Electrotransfection efficiency | Cell survival rate | |
| Apparatus of the present invention | 88.6% | 98.1% |
| Electricity revolving cup in the prior art | 78.3% | 89.1% |
Although the present invention is described in detail, modification within the spirit and scope of the present invention is for those skilled in the art
Member will be apparent.It should be understood that described above and/or aspect of the invention in the appended claims and
The part of various embodiments and various features can be combined or completely or partially be interchangeable.Such as those skilled in the art
It will be realized that referring to those of another embodiment embodiment can be suitably in the description of aforementioned various embodiments
With other combination of embodiment.In addition, it will be recognized by those of ordinary skill in the art that foregoing description is only as an example, and unexpectedly
The present invention to be limited.
Claims (10)
1. a kind of vertical intermittent formula streaming electrotransfection device, including cavity, first electrode, second electrode, the first conduit, second is led
Pipe；It is characterized in that, the first electrode and second electrode are respectively placed in the symmetrical wall of cavity, and first electrode and the second electricity
Pole can be electrically connected by the side wall of cavity with outside formation；Inlet and liquid outlet be set on the cavity wall, it is described into
Liquid mouth and the first conduit are tightly connected, and the liquid outlet and the second conduit are tightly connected.
2. a kind of vertical intermittent formula streaming electrotransfection device according to claim 1, which is characterized in that the first electrode
It is placed in the top of cavity, the second electrode is placed in the bottom of cavity.
3. a kind of vertical intermittent formula streaming electrotransfection device according to claim 1, which is characterized in that the first electrode
It is placed in the left side of cavity, the second electrode is placed in the right side of cavity.
4. a kind of vertical intermittent formula streaming electrotransfection device according to claim 2 or 3, which is characterized in that the feed liquor
Mouth is set to the bottom of cavity wall.
5. a kind of vertical intermittent formula streaming electrotransfection device according to claim 2 or 3, which is characterized in that the liquid out
Mouth is set to the top of cavity wall.
6. a kind of vertical intermittent formula streaming electrotransfection device according to claim 1-3, which is characterized in that described
One-way control valve is set on first conduit.
7. a kind of vertical intermittent formula streaming electrotransfection device according to claim 1-3, which is characterized in that described
One-way control valve is set on second conduit.
8. a kind of vertical intermittent formula streaming electrotransfection device according to claim 1-7, which is characterized in that described
The device that containment portion setting mixes intracavity liquid.
9. a kind of vertical intermittent formula streaming electrotransfection device according to claim 8, which is characterized in that described device is also wrapped
Include pump.
10. a kind of vertical intermittent formula streaming electrotransfection device according to claim 8, which is characterized in that the cavity is adopted
It is prepared with the mode that mold pours, makes the first electrode and second electrode i.e. embeddable cavity during cavity preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810493935.6A CN110527624A (en) | 2018-05-24 | 2018-05-24 | A kind of intermittent streaming electrotransfection device |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810493935.6A CN110527624A (en) | 2018-05-24 | 2018-05-24 | A kind of intermittent streaming electrotransfection device |
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| CN110527624A true CN110527624A (en) | 2019-12-03 |
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| CN201810493935.6A Pending CN110527624A (en) | 2018-05-24 | 2018-05-24 | A kind of intermittent streaming electrotransfection device |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3862420A1 (en) * | 2020-02-07 | 2021-08-11 | Bühler AG | Modified treatment chamber for treating cells |
| WO2024012261A1 (en) * | 2022-07-14 | 2024-01-18 | 苏州壹达生物科技有限公司 | Flow electroporation device |
-
2018
- 2018-05-24 CN CN201810493935.6A patent/CN110527624A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3862420A1 (en) * | 2020-02-07 | 2021-08-11 | Bühler AG | Modified treatment chamber for treating cells |
| WO2021156373A1 (en) * | 2020-02-07 | 2021-08-12 | Bühler AG | Modified treatment chamber for treating cells |
| WO2024012261A1 (en) * | 2022-07-14 | 2024-01-18 | 苏州壹达生物科技有限公司 | Flow electroporation device |
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| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: No.2 501, No.200, Shengpu Xingpu Road, Suzhou Industrial Park, Jiangsu Province Applicant after: SUZHOU ETTA BIOTECH CO., LTD. Address before: 215123 Ruoshui Road 388 E1504, Suzhou Industrial Park, Jiangsu Province Applicant before: SUZHOU ETTA BIOTECH CO., LTD. |
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| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20191203 |