CN106995783A - Cell electrotransfection device and its application - Google Patents
Cell electrotransfection device and its application Download PDFInfo
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- CN106995783A CN106995783A CN201610044870.8A CN201610044870A CN106995783A CN 106995783 A CN106995783 A CN 106995783A CN 201610044870 A CN201610044870 A CN 201610044870A CN 106995783 A CN106995783 A CN 106995783A
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Abstract
The invention belongs to cell transfection technique field, and in particular to a kind of cell electrotransfection device and its application.The cell electrotransfection device, at least include an electrotransfection control circuit, two electrodes and an electrotransfection container, two electrodes control the power supply in circuit to be connected with electrotransfection, one of electrode is used as positive electrode, another electrode is used as negative electrode, two electrodes are in the electrotransfection container, and the surface of one or two electrode in described two electrodes has nano thread structure.The cell electrotransfection device of the present invention, can be used in the transfection of various kinds of cell, and cell survival rate is high, and transfection is good, possesses very high application value.
Description
Technical field
The invention belongs to cell transfection technique field, and in particular to a kind of cell electrotransfection device and its application.
Background technology
Cell transfecting is that exogenous DNA, RNA, albumen, polypeptide, micromolecular compound etc. are imported into one kind of cell specially
Gate technique, can be applied to eukaryotic, bacterium.With continuing to develop for molecular biology and RESEARCH ON CELL-BIOLOGY, transfect
Have become the conventional tool of research and control cytogene function.In research gene function, controlling gene expression, mutation point
In the biological test such as analysis and protein production, its application is more and more extensive.
Cellular immunity based on Chimeric antigen receptor (CAR) turns into the emerging form for the treatment of malignant tumour at present.CAR is repaiied
Decorations T cell is will to recognize that the single-chain antibody of tumor associated antigen and the activation motifs of T cell are combined as a whole, i.e., by antibody pair
The high-affinity of tumour antigen is combined with the killing mechanism of T lymphocytes, by gene transfer method transfecting T cells, is made
It has specific recognition and the ability of killing tumor cell.
CAR-T immunotherapies are that T cell is isolated from blood samples of patients, and then it is carried out after genetic modification, makes T cell
Surface expression Chimeric antigen receptor.Fed back to after being expanded in laboratory to these T cells in patient's body, utilize these T
Cells against tumor cells carry out accurate immune attack.
Therefore, the immunocyte of CAR modifications needs gene transfer method efficiently, safe.Clinically it is used for carrying CAR at present
Carrier be mainly derived from retrovirus and slow virus.The application of wherein retrovirus is more ripe, passes through film in virus
The acceptor interaction of glycoprotein and host cell surface and enter host cell, afterwards reverse transcriptase start synthetic DNA and with
Machine is incorporated into host genome, being capable of stable transfection particular host cell.But this method can only transfect the cell of division stage,
And there is certain potential safety hazard：First：The problem of there is radom insertion integration due to transcription vector, can be to patient certainly
Body cell causes irreversible genetic manipulation, there is the potential risk for producing canceration；Second：Current CAR-T cells are usually to use
Reverse transcription or the periphery blood T lymphocyte of slow-virus transfection, screen and amplify stable expression cell in vitro.Work as treatment
Complete or these transgenic T cells how are disposed when there is side effect as a future trouble problem；3rd：Viral vector
Transgenic technology is operationally cumbersome, can cause the unfavorable factor problem for producing quality management and control.Safety problem turns into disease
The inherent shortcoming of poisonous carrier transgenosis.
Non-virus carrier transgenic method common are lipofection and electrotransfection method.Lipofection is by transfection efficiency institute
Limit, is not suitable for clinical practice at present.
Electrotransfection is then to simultaneously form the micropore that nucleic acid can be allowed to pass through on cell membrane by DC Electric Field, and nucleic acid is existed
Actively enter cell under electric field action.The commercialization electroporation apparatus being widely used at present, more using parallel aluminum electrode,
Need up to more than 200V voltage and transfection reagent box to aid in, stronger fuel factor and electrochemistry can be produced under action of high voltage
Effect, metal electrode produces metal cation in electrochemical reaction and toxicity is produced to cell, and the electric field that cell is subject to is made
The factors such as inhomogeneities cause the death rate of cell higher, transfection heterogeneity.
The Gene Pulser MXcell electroporations output waveform of Bio-Rad companies is exponential wave or square wave, the transfection of most cells
Voltage is 200-400V, and needs to use supporting electroporation buffer.
The Neon electroporation output waveforms of Life technology companies are only square wave, output voltage 500-2500V, most cells
Transfection voltage be more than 1000V, and need using corresponding electricity to turn buffer solution according to the difference of cell type.
In recent years, nano material electrode is also applied to intracellular drug delivery.The silicon nano-array material of PEI modifications is used to turn
Mammalian cell is contaminated, but relies solely on penetration of the nano wire to cell membrane, transfection efficiency is than relatively low.
Nano material is used as to the electrode of electrotransfection, in the presence of extra electric field power, higher transfection efficiency can be produced.Receive
Rice material electrodes are compared with ordinary electrode, and its special electrode surface microstructure can make local field strength increase by 3~4 quantity
Level, so that applied voltage is substantially reduced, its nanoscale preferably can interact with cell in addition, can overcome biography
System electrotransfection technology high voltage and the low limitation of cell survival rate, thus with very big application value.
Retrieval to prior art finds that Xiexi et al. exists《ACSNANO》On delivered an entitled " Nanostraw
Electroporation System for Highly Efficient Intracellular Delivery and Transfection " article, should
Text is prepared for aluminum oxide using the method for ald and reactive ion etching in a kind of porous polycarbonate film of commercialization
Nanotube, tube diameters 250nm, 1.5 μm of length, in voltage 6-20V, pulsewidth 20-200 μ s, 200-2000 pulse
Under conditions of cell survival rate 90%, transfection efficiency reaches 70%.However, this method is still main to utilize nanotube to adherent thin
The mechanism of born of the same parents, the electrophoretic action of electric field is transfected.And the suspension cell of small volume can not be attached to nanotube substrate
Surface, and can not be transfected with the device.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide a kind of based on nano line electrode
Cell electrotransfection device and its application.
To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：
The first aspect of the present invention is there is provided a kind of cell electrotransfection device, at least including electrotransfection control circuit, two electricity
Pole and an electrotransfection container, two electrodes control the power supply in circuit to be connected with electrotransfection, and one of electrode is used as positive electricity
Pole, another electrode as negative electrode, two electrodes in the electrotransfection container, one in described two electrodes or
The surface of two electrodes has nano thread structure.
Preferably, the nano wire is selected from cupric oxide nano line, titanium dioxide nano thread, nanowires of gold, nano silver wire, copper
Nano wire, titanium nano wire, Fe nanowire, Pt nanowires, titanium platinum alloy nano wire, iron oxide nano-wire, titanium dioxide sijna
Any of rice noodles, silicon nanowires of metallic cover.
When the material of only one of which electrode in two electrodes is nano wire, the nano wire can directly be selected from above-mentioned each nano wire
Any of.
When the material of two electrodes is nano wire, the nano-material of two electrodes be able to can also be differed with identical.
Preferably, the direction of growth of the nano wire is consistent.
Preferably, the nano wire grows perpendicular to substrate surface.
In the present invention, when the diameter of nano wire is in 1nm~5 μ m, referred to as it is nano wire, is not limited to
The diameter of nano wire must be in below 100nm.
Preferably, the diameter range of the nano wire is：30nm~5 μm.It is further preferred that the diameter range of the nano wire is：
50nm~1 μm.
Preferably, the length range of the nano wire is 3 μm~300 μm.It is further preferred that the length range of the nano wire is at 10 μm
Between~100 μm.
It is further preferred that the nano wire is selected from CuO nano wires, the diameter range of the CuO nano wires exists
Between 50nm~150nm, length range is between 10 μm~20 μm.
It is further preferred that the nano wire is selected from titanium dioxide nano thread, the diameter range of the titanium dioxide nano thread exists
Between 150nm~200nm, length range is between 5 μm~100 μm.
It is further preferred that the nano wire is selected from nanowires of gold, the diameter range of the nanowires of gold is in 100nm~150nm
Between, length range is between 10 μm~15 μm.
Preferably, two electrodes are fixed in electrotransfection container by electrode fixed mount.
Preferably, the distance between two electrodes scope is 0.5mm~5mm.
Preferably, the electrotransfection control circuit can provide the transfection waveform such as square wave, pulse, sine wave.For example, can be using letter
Number general source DG1022U of generator is used as electrotransfection power supply.
Preferably, the electrotransfection container is shaped as any of tubulose, cylindrical type, cuboid, polygonal.
Nano wire in foregoing electrotransfection device can be prepared using method of the prior art, for example：Thermal oxidation method, template,
Chemical vapour deposition technique.
The second aspect of the present invention is including as follows there is provided a kind of method that electrotransfection is carried out using foregoing electrotransfection device
Step：
(1) in electrotransfection container, cell to be transfected and material to be transfected are added；
(2) electrotransfection control circuit is opened, electrotransfection is carried out.
Preferably, foregoing electrotransfection device is used to carry out electrotransfection method of the method for electrotransfection for non-treatment diagnostic purpose.
Preferably, in step (1), the cell to be transfected is selected from suspension cell or attached cell.It is further preferred that
The cell to be transfected is selected from stem cell, DC cells, macrophage, lymphocyte.
Preferably, in step (1), the density range of the cell to be transfected is 5 × 105Individual/ml~5 × 107Individual/ml.
Preferably, in step (1), the cell to be transfected is in static or flow regime.It is further preferred that working as
When the cell to be transfected is in flow regime, flowing velocity is 300L/ (hm2)~900L/ (hm2)。
Preferably, in step (1), the material to be transfected be selected from DNA, siRNA, microRNA, albumen,
Polypeptide.
Preferably, in step (2), the electrotransfection control circuit uses signal generator.It is further preferred that institute
Signal generator is stated from general source DG1022U.
Preferably, third aspect present invention there is provided aforementioned cells electrotransfection device in cell electrotransfection instrument is prepared
Purposes.
Compared with prior art, the present invention has the advantages that：
The present invention constructs the cell electrotransfection device based on nano line electrode first by in-depth study extensively.The present invention
Cell electrotransfection device, used transfection voltage be less than or equal to 10V, applicable cell type be include various kinds of cell system and
Various adherent and suspension cell including primary cell.That is, under conventional low-voltage, you can realize that effect is up to 200,000 RLU
Electrotransfection, and Transfected cells survival rate is above more than 60%.
In summary, cell electrotransfection device manufacture craft of the invention is simple, with low cost, in use, whole transfection process
It is simple to operation, can be used in the transfection of various kinds of cell, and cell survival rate is high, transfection is good, possess it is very high should
With value.
Brief description of the drawings
Fig. 1：Outside drawing before and after copper mesh oxidation in the embodiment of the present invention.
Fig. 2：CuO nano wires SEM schemes in the embodiment of the present invention.
Fig. 3：Electrotransfection electrode schematic diagram.
Fig. 4：Electrotransfection electrode fixed mount schematic diagram.
Fig. 5：Electrotransfection schematic device.
Fig. 6 A：The cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 is for the suitable of Chinese hamster ovary celI
Suitable transfection conditions are：Voltage 8V, pulsewidth 30ms, cycle 1s, pulse number 20 times.
Fig. 6 B：Using the cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 and for CHO
The suitable transfection conditions of cell：Voltage 8V, pulsewidth 30ms, cycle 1s, pulse number 20 times, respectively using Opti-MEM,
Hepes buffer solutions (10mM HEPES, 272mM sucrose, pH7.3), sucrose phosphate buffer (10mM phosphate,
272mM sucrose, pH7.3) electrotransfection is carried out, illustrate, during electrotransfection Chinese hamster ovary celI, buffer solution is turned using Opti-MEM
Contaminate effect preferable.
Fig. 7：The cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 is for RAW264.7 cells
Suitably transfection conditions are：Voltage 8V, pulsewidth 10ms, cycle 1s, pulse number 8 times.
Fig. 8：The cell electrotransfection device containing titanium dioxide nano line electrode constructed by embodiment 5 is for Chinese hamster ovary celI
Suitably transfection conditions are：Voltage 8V, pulsewidth 30ms, cycle 1s, pulse number 15 times.
Fig. 9：The cell electrotransfection device containing titanium dioxide nano line electrode constructed by embodiment 5 is for Jurkat cell
Suitably transfection conditions are：Voltage 10V, pulsewidth 10ms, cycle 1s, pulse number 14 times.
Figure 10：The cell electrotransfection device containing nanowires of gold electrode constructed by embodiment 6 turns for the suitable of Chinese hamster ovary celI
Dye condition is：Voltage 8V, pulsewidth 30ms, cycle 1s, pulse number 20 times.
Figure 11：The cell electrotransfection device containing nanowires of gold electrode constructed by embodiment 6 is for the suitable of Jurkat cell
Transfection conditions are：Voltage 8V, pulsewidth 10ms, cycle 1s, pulse number 16 times.
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to following spies
Fixed specific embodiment；It is also understood that the term used in the embodiment of the present invention is to describe specific specific implementation
Scheme, the protection domain being not intended to be limiting of the invention.The test method of unreceipted actual conditions in the following example, leads to
Often according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two end points of each number range with
And any one numerical value can select between two end points.Unless otherwise defined, all technologies used in the present invention and section are academic
Language is identical with the meaning that those skilled in the art of the present technique are generally understood that.In addition to the specific method used in embodiment, equipment, material,
According to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and the embodiment of the present invention
Described in method, any method, equipment and the material of equipment, material similar or equivalent prior art realize the present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method use the art
Conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of the CuO nano line electrodes of embodiment 1
The present embodiment prepares CuO nano line electrodes using thermal oxidation method, specifically includes following steps：
Purity is more than 99.9%, string diameter 0.04mm, aperture 0.09mm, 200 mesh square hole copper mesh of plain weave are washed into institute
Shape is needed, and is flattened with tablet press machine.Copper mesh is placed in 1.0molL-1Dilute hydrochloric acid solution in soak about 30min, to dissolve table
The oxide and impurity in face.Rinsed, untill washing lotion is neutrality, finally, dried up with nitrogen repeatedly with deionized water again.Will
Copper mesh after cleaning-drying is put into quartz boat, in feeding ramped heating schedule stove, in 0.03MPa atmospheric atmosphere, with 5
DEG C/min heating rate raises temperature to 500 DEG C, then after 500 DEG C of heating 2h, cools to room temperature with the furnace.After reaction terminates
It can be seen that copper mesh surface is covered by the oxide layer of one layer of black.Outward appearance is as shown in Figure 1 before and after copper mesh oxidation.By copper mesh thermal oxide
The CuO nano wires that method is prepared, are observed in the secure execution mode (sem, as a result as shown in Fig. 2 the CuO nanowire growth directions
Unanimously, perpendicular to copper-based growth, bottom is thick, top is sharp.The diameter range of the CuO nano wires between 50~150nm,
Length is at 10~20 μm.In addition, CuO nano wires can be also prepared using existing other technologies, as long as prepared CuO
Nanometer line morphology, diameter and length meet the requirements.
The structure of the cell electrotransfection device of embodiment 2
Using CuO nano wires prepared in embodiment 1 as electrode (as shown in Figure 3), cell electrotransfection device is built.
The electrotransfection device, at least holds including an electrotransfection control circuit, two CuO nano line electrodes and an electrotransfection
Device.The electrotransfection control circuit can use prior art, and such as signal generator (general source DG1022U) can be produced
Accurately, stably, the output signal of low distortion, include five kinds of reference waveforms such as square wave, pulse, sine wave, and using appointing
Waveform required for Yi Bo softwares for editing Ultrawave outputs, output voltage range 2mV~10V, frequency range
1 μ Hz~25MHz；The ginsengs such as waveform, voltage, pulsewidth, pulse number that can be according to required for electricity turns the different flexible modulations of cell
Number.Two CuO nano line electrodes control the power supply in circuit to be connected with electrotransfection.Two CuO nano line electrodes can be located at electricity
Transfect in container, and it is (as shown in Figure 4) fixed by electrode fixed mount.One of electrode can as positive pole, in addition one
It is individual can be as negative pole, after energization, you can formed electric field, be opposite to electrotransfection container cell carry out electrotransfection.
As shown in figure 5, the building process of electrotransfection device, can include step：
On the first step, the platform that CuO nano line electrodes 2 are placed on to fixed mount bottom, blend rubber circle is compressed；Second step,
By CuO nano line electrodes 1 be placed on cell suspension container (that is, electrotransfection container can for example use the hole of commercialization 24
Culture plate) bottom；3rd step, fixed mount is inserted in cell suspension container, and electrode 1 is compressed；Electrode fixed mount
Bottom platform highly be used to control the spacing of parallel pole 1 and electrode 2 in 1~2mm；4th step, by CuO nano wires electricity
Pole 1 and CuO nano line electrodes 2 are respectively adopted the wire with crocodile clip and are connected with signal generator (general source DG1022U),
And voltage is applied by signal generator；5th step：Cell suspension is added in cell suspension container can carry out electricity turn operation.
The cell electrotransfection device of embodiment 3 transfects attached cell
Attached cell, this implementation are transfected with the cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2
Example explanation by taking Chinese hamster ovary celI as an example.
Transfection method comprises the following steps：
(1) electroporation (electrotransfection) the previous day, with proper density passage cell, cell is made to be in exponential phase before transfection,
Cell monolayer degrees of fusion about 70-80%；
(2) two CuO nano line electrodes, electrode fixed mount and packing ring are placed in 75% alcohol and soaked 2 hours, Ran Hou
Irradiate after 30min and can be used under uviol lamp, CuO nano line electrodes and electricity are placed in the way of in embodiment 2
Pole fixed mount；
1ml DMEM culture mediums (containing 10% serum, without antibiotic) are added in (3) 6 porocyte culture plates per hole, are placed on
37 DEG C of CO2Preheated in incubator；
(4) the logarithmic phase cell of culture is taken out, is discarded after culture medium, PBS washing cells, trypsin digestion cell 2min treats thin
After born of the same parents are rounded, are terminated and digested with DMEM complete mediums；
(5) cell is gently blown down into after single cell suspension, 1000rpm room temperatures centrifugation 5min；
(6) supernatant discarding, addition PBS resuspension cells, counting after piping and druming is uniform, and according to cell density, take certain body completely
Long-pending re-suspension liquid；
(7) 1000rpm room temperatures centrifugation 5min, complete supernatant discarding, add Opti-MEM culture mediums and are resuspended and blow and beat uniformly,
Addition treats that Pignus pignoris grain pGL3 is mixed, and it is 5 × 10 to make final cell densities6Individual/ml, the final concentration of 0.1mg/ml of plasmid；Up and down
Soft piping and druming is uniform；
(8) 250 μ l cell suspensions are added into the cell culture plate well for assemble electrode；The both positive and negative polarity of signal generator is connected
On two plate electrodes (electrode 2 and electrode 1) up and down, apply voltage；Electricity turns condition：Voltage 8V, pulsewidth 50ms,
Frequency 1Hz, pulse number 10 times；And turn condition using other electricity as shown in Figure 6, carry out electrotransfection；
(9) cell suspension after electric shock is added in the culture medium of preheating, after blowing and beating uniformly up and down, puts back to 37 DEG C of CO2Training
Support and normally cultivated in case；Electricity carries out cell survival rate detection immediately after turning；And after transfection 24h, carry out Luciferase
Detection of expression.
Cell survival rate detection method：
In (1) 96 orifice plate, 90 μ l culture mediums are previously added per hole；
(2) after electricity turns, take 10 μ l electricity to turn cell suspension, be added in 96 orifice plates；
(3) then, under the conditions of lucifuge, 10 μ l CCK8 reaction substrates are added per hole；
It is incubated in (4) 37 DEG C of cell culture incubators after 1-4h, the absorbance in each hole at 450nm wavelength is determined with ELIASA；
(5) cell survival rate (%)=[A (electricity turns)-A (blank)]/[A (non-electrical turns)-A (blank)] × 100
A (electricity turns)：With CCK8 solution, turn the absorbance in the cell suspension hole of processing through electricity；
A (non-electrical turns)：With CCK8 solution, the cell suspension hole that electricity turn processing is not done (is used and electric turn hole
Identical buffer solution suspension cell) absorbance；
A (blank)：Turn absorbance of the buffer solution without the hole of cell with CCK8 solution, electricity.
Luciferase detection of expression methods：
(1) cell transfecting discards culture medium after 24 hours, and PBS is washed one time；
(2) 5 × cell pyrolysis liquid (Promega) is diluted to 1 with pure water ×, and recover using preceding to room temperature；
(3) 150 μ l1 × cell pyrolysis liquid is added per hole and is rocked, it is fully covered cell, freeze thawing in -30 degree refrigerators is put into
30min；
(4) cell is scraped from plate bottom, and all liq is transferred in centrifuge tube, 12,000g centrifugations take supernatant；
(5) the 50 above-mentioned lysates of μ l and 20 μ l Luciferase are determined into liquid to mix；
(6) it is immediately placed in fluorescence illumination photometer and determines, stand-by period 0.6s, time of measuring 10s.
Cell Transfection Conditions optimize：
The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in Figure 6A：Constructed by embodiment 2
The cell electrotransfection device containing CuO nano line electrodes be for the suitable transfection conditions of Chinese hamster ovary celI：Voltage 8V, arteries and veins
Wide 30ms, cycle 1s, pulse number 20 times.
In addition, the cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 carries out electricity for Chinese hamster ovary celI
Transfection, under each transfection conditions as shown in Figure 6A, cell survival rate is above 60%.
Using the cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 and for the suitable of Chinese hamster ovary celI
Suitable transfection conditions：Voltage 8V, pulsewidth 30ms, cycle 1s, pulse number 20 times, respectively using Opti-MEM, Hepes
Buffer solution (10mM HEPES, 272mM sucrose, pH7.3), sucrose phosphate buffer (10mM phosphate, 272mM
Sucrose, pH7.3) electrotransfection is carried out, as a result as shown in Figure 6B, during electrotransfection Chinese hamster ovary celI, buffer solution uses Opti-MEM
Transfection is preferable, up to 200,000 RLU.
The cell electrotransfection device Transfection of macrophages of embodiment 4
With the cell electrotransfection device Transfection of macrophages containing CuO nano line electrodes constructed by embodiment 2, this implementation
Example explanation by taking RAW264.7 cells as an example.
Transfection procedure is as follows：
(1) electroporation the previous day, with proper density passage cell, cell is made to be in exponential phase, cell monolayer before transfection
Degrees of fusion about 70-80%；
(2) electrode, fixed mount and packing ring are placed in 75% alcohol and soaked 2 hours, then irradiated under uviol lamp after 30min
It can be used, electrode and fixed mount placed in the way of in embodiment 2；
1ml RPMI-1640 culture mediums are added per hole (contain 10% serum, 20ng/ml GM-CSF, without anti-in (3) 6 orifice plates
Raw element), it is placed in 37 DEG C of CO2 incubators and preheats.
(4) the logarithmic phase cell of culture is taken out, culture medium is discarded, is blown and beaten cell from culture dish bottom with the PBS of precooling.
(5) cell is blown and beaten into uniform rear counting, and according to cell density, takes the cell suspension of certain volume.
(6) 1000rpm room temperatures centrifugation 5min, complete supernatant discarding, add Opti-MEM culture mediums and are resuspended and blow and beat uniformly,
Addition treats that Pignus pignoris grain pGL3 is mixed, and it is 10 to make final cell densities7Individual/ml, the final concentration of 0.1mg/ml of plasmid.It is light up and down
Soft piping and druming is uniform.
(7) into the cell culture plate well for assembling electrode, 250 μ l cell suspensions are added per hole；By the positive and negative of signal generator
Pole be connected to above and below on two plate electrodes (electrode 2 and electrode 1), apply voltage；Electricity turns condition：Voltage 8V, pulsewidth
10ms, frequency 1Hz, pulse number 8 times；And turn condition using other electricity as shown in Figure 7, carry out electrotransfection；
(8) cell suspension after electric shock is added in the culture medium of preheating, after piping and druming is uniform, puts back to 37 DEG C of CO2Incubator
In normally cultivate；Electricity carries out cell survival rate detection immediately after turning；And after transfection 24h, carry out Luciferase expression
Detection.
Cell survival rate detection method：Cell survival rate detection method described in reference implementation example 3.
Luciferase detection of expression methods：Luciferase detection of expression method described in reference implementation example 3.
Cell Transfection Conditions optimize：
The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in Figure 7：Constructed by embodiment 2
Cell electrotransfection device containing CuO nano line electrodes is for the suitable transfection conditions of RAW264.7 cells：Voltage 8V,
Pulsewidth 10ms, cycle 1s, pulse number 8 times.
In addition, the cell electrotransfection device containing CuO nano line electrodes constructed by embodiment 2 is directed to RAW264.7 cells
Electrotransfection is carried out, under each transfection conditions as shown in Figure 7, cell survival rate is above 60%.
The preparation of the titanium dioxide nano line electrode of embodiment 5, structure, the cell transfecting of cell electrotransfection device
The present embodiment prepares titanium dioxide nano line electrode using thermal oxidation method, specifically included with reference to the preparation method of embodiment 1
Following steps：
The titanium net cleaned up is placed in ramped heating schedule stove, in 0.03MPa atmospheric atmosphere, with 5 DEG C/min's
Heating rate raises temperature to 750 DEG C, then after 750 DEG C of heating 10h, cools to room temperature with the furnace.The titanium dioxide prepared
Titanium nano wire is perpendicular to substrate surface, and the direction of growth is consistent, and diameter range is between 150~200nm, and length is about 5~100 μm.
In addition, also titanium dioxide nano thread can be prepared using existing other technologies, as long as prepared titanium dioxide nano thread
Form, diameter and length meet the requirements.
With reference to the method for embodiment 2, using the titanium dioxide nano thread prepared by the present embodiment as electrode, build cell electricity and turn
Contaminate device.
The electrotransfection device, at least including an electrotransfection control circuit, two titanium dioxide nano line electrodes and an electricity
Transfect container.The electrotransfection control circuit can use prior art, such as signal generator (general source DG1022U), energy
It is enough produce it is accurate, stably, the output signal of low distortion, include five kinds of reference waveforms such as sine wave, square wave, pulse, and can
Waveform required for being exported using any ripple software for editing Ultrawave, output voltage range 2mV~10V, frequency range
1 μ Hz~25MHz；The ginsengs such as waveform, voltage, pulsewidth, pulse number that can be according to required for electricity turns the different flexible modulations of cell
Number.Two titanium dioxide nano line electrodes control the power supply in circuit to be connected with electrotransfection.Two titanium dioxide nano line electrodes
Can be in electrotransfection container, and fixed by electrode fixed mount.One of electrode can be as positive pole, and another can make
For negative pole, after energization, you can form electric field, the cell for being opposite to electrotransfection container carries out electrotransfection.
Attached cell is transfected with the cell electrotransfection device containing titanium dioxide nano line electrode constructed by the present embodiment 5, this
Embodiment explanation by taking Chinese hamster ovary celI as an example.Specific transfection method, cell survival rate detection method and Luciferase expression
Detection method is referring to embodiment 3.
The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in Figure 8：Constructed by embodiment 5
Cell electrotransfection device containing titanium dioxide nano line electrode is for the suitable transfection conditions of Chinese hamster ovary celI：Voltage 8V,
Pulsewidth 30ms, cycle 1s, pulse number 15 times.
In addition, the cell electrotransfection device containing titanium dioxide nano line electrode constructed by embodiment 5 enters for Chinese hamster ovary celI
Row electrotransfection, under each transfection conditions as shown in Figure 8, cell survival rate is above 60%.
Lymphocyte is transfected with the cell electrotransfection device containing titanium dioxide nano line electrode constructed by the present embodiment 5, this
Embodiment explanation by taking suspension cell Jurkat as an example.
Transfection procedure is as follows：
Electroporation the previous day, with proper density passage cell, cell is set to be in exponential phase before transfection.
(1) electrode, fixed mount and packing ring are placed in 75% alcohol and soaked 2 hours, then irradiated under uviol lamp after 30min
It can be used；The mode as described in embodiment 2 places electrode and fixed mount；
1ml RPMI-1640 culture mediums (containing 10% serum, without antibiotic) are added in (2) 12 orifice plates per hole, 37 are placed on
DEG C CO2Preheated in incubator；
(3) the logarithmic phase cell of culture is taken out, 1000rpm room temperatures centrifugation 5min discards culture medium；
(4) add PBS and cell is resuspended, counted after piping and druming is uniform, and according to cell density, take the re-suspension liquid of certain volume；
(5) 1000rpm room temperatures centrifugation 5min, complete supernatant discarding, add Opti-MEM culture mediums and are resuspended and blow and beat uniformly,
Addition treats that Pignus pignoris grain pGL3 is mixed, and it is 10 to make final cell densities7Individual/ml, the final concentration of 0.1mg/ml of plasmid.Up and down
Soft piping and druming is uniform；
(6) 250 μ l cell suspensions are added per hole；By the both positive and negative polarity of signal generator be connected to above and below on two plate electrodes, apply electricity
Pressure.Electricity turns condition：Voltage 8V, pulsewidth 10ms, frequency 1Hz, pulse number 8 times；
(7) cell suspension after electric shock is added in the culture medium of preheating, after piping and druming is uniform, puts back to 37 DEG C of CO2Incubator
In normally cultivate；Electricity carries out cell survival rate detection immediately after turning；And after transfection 24h, carry out Luciferase tables
Up to detection.
Specific cell survival rate detection method and Luciferase detection of expression method are referring to embodiment 3.
The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in Figure 9：Constructed by embodiment 5
Cell electrotransfection device containing titanium dioxide nano line electrode is for the suitable transfection conditions of Jurkat cell：Voltage 10V,
Pulsewidth 10ms, cycle 1s, pulse number 14 times.
In addition, the cell electrotransfection device containing titanium dioxide nano line electrode constructed by embodiment 5 enters for Jurkat cell
Row electrotransfection, under each transfection conditions as shown in Figure 9, cell survival rate is above 60%.
The preparation of the nanowires of gold electrode of embodiment 6, structure, the cell transfecting of cell electrotransfection device
The present embodiment prepares nanowires of gold electrode using template, specifically includes following steps：
Purity is more than 99.9%, thickness is placed in 1molL for 10 μm of goldleaf-1Dilution heat of sulfuric acid in be cleaned by ultrasonic 30min.
It is template using polycarbonate membrane, nanowires of gold is prepared by electrochemical deposition method.Wherein, anode electrode is platinized platinum, negative electrode
For the goldleaf cleaned in advance and makrolon perforated membrane (aperture：100 nanometers；Film thickness：6 millimeters；Hole density 107cm2),
It is containing 0.5M HClO that makrolon perforated membrane, which is tightly covered in electrolyte component in goldleaf surface, electrolytic cell,4Gold chloride it is molten
Liquid (w/w 1%), carries out electro-deposition under 0.18V constant potential.After deposition terminates, template is immersed in 4 DEG C of CHCl3
2h dissolves template molecule in solution, that is, obtains nanowires of gold electrode.According to the aperture of polycarbonate membrane, diameter is obtained
100~150nm nanowires of gold electrode.Sedimentation time 15min is controlled, nanowire length is about 10~15 μm.
In addition, also nanowires of gold can be prepared using existing other technologies, if prepared nanowires of gold form, diameter with
Length meets the requirements.
With reference to the method for embodiment 2, using the nanowires of gold prepared by the present embodiment as electrode, cell electrotransfection device is built.
The electrotransfection device, at least holds including an electrotransfection control circuit, two nanowires of gold electrodes and an electrotransfection
Device.The electrotransfection control circuit can use prior art, and such as signal generator (general source DG1022U) can be produced
Accurately, stably, the output signal of low distortion, include five kinds of reference waveforms such as sine wave, square wave, pulse, and using appointing
Waveform required for Yi Bo softwares for editing Ultrawave outputs, output voltage range 2mV~10V, frequency range
1 μ Hz~25MHz；The ginsengs such as waveform, voltage, pulsewidth, pulse number that can be according to required for electricity turns the different flexible modulations of cell
Number.Two nanowires of gold electrodes control the power supply in circuit to be connected with electrotransfection.Two nanowires of gold electrodes can be located at electrotransfection
In container, and fixed by electrode fixed mount.One of electrode can be as positive pole, and another can be powered as negative pole
Afterwards, you can form electric field, the cell for being opposite to electrotransfection container carries out electrotransfection.
Attached cell, the present embodiment are transfected with the cell electrotransfection device containing nanowires of gold electrode constructed by the present embodiment 6
The explanation by taking Chinese hamster ovary celI as an example.Specific transfection method, cell survival rate detection method and Luciferase detection of expression sides
Method is referring to embodiment 3.The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in Figure 10：It is real
Apply the cell electrotransfection device containing nanowires of gold electrode constructed by example 6 is for the suitable transfection conditions of Chinese hamster ovary celI：Electricity
Press 8V, pulsewidth 30ms, cycle 1s, pulse number 20 times.
In addition, the cell electrotransfection device containing nanowires of gold electrode constructed by embodiment 6 carries out electricity for Chinese hamster ovary celI and turned
Dye, under each transfection conditions as shown in Figure 10, cell survival rate is above 60%.
Lymphocyte, the present embodiment are transfected with the cell electrotransfection device containing nanowires of gold electrode constructed by the present embodiment 6
The explanation by taking suspension cell Jurkat as an example.
The conditions such as voltage, pulsewidth, cycle, pulse number are optimized, as a result as shown in figure 11：Constructed by embodiment 6
The cell electrotransfection device containing nanowires of gold electrode be for the suitable transfection conditions of Jurkat cell：Voltage 8V, pulsewidth
10ms, cycle 1s, pulse number 16 times.
In addition, the cell electrotransfection device containing nanowires of gold electrode constructed by embodiment 6 carries out electricity for Jurkat cell and turned
Dye, under each transfection conditions as shown in figure 11, cell survival rate is above 60%.
The present inventor is also closed using nano silver wire, copper nano-wire, titanium nano wire, Fe nanowire, Pt nanowires, titanium platinum
Nanowires of gold, iron oxide nano-wire, stannic oxide nano wire, metallic cover silicon nanowires as electrode cell electrotransfection
Device, the diameter range of each nano wire is：30nm~5 μm；Length range is between 3 μm~300 μm；It is preferred that respectively receiving
The diameter range of rice noodles is：50nm~1 μm；Length range is between 10 μm~100 μm.Through experiment, for various patches
Parietal cell carries out electrotransfection, can obtain the cell survival rate and transfection similar with foregoing three kinds of electrotransfection devices.
It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, should
Point out, for those skilled in the art, on the premise of the inventive method is not departed from, can also make some
Improve and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, not
In the case of departing from the spirit and scope of the present invention, when a little change made using disclosed above technology contents, repair
Decorations and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile, all substantial technologicals according to the present invention are to above-mentioned reality
The variation, modification and evolution for any equivalent variations that example is made are applied, in the range of still falling within technical scheme.
Claims (17)
1. a kind of cell electrotransfection device, at least including an electrotransfection control circuit, two electrodes and an electrotransfection container,
Two electrodes control the power supply in circuit to be connected with electrotransfection, and one of electrode is made as positive electrode, another electrode
For negative electrode, two electrodes are in the electrotransfection container, the surface of one or two electrode in described two electrodes
With nano thread structure.
2. cell electrotransfection device according to claim 1, it is characterised in that the nano wire be selected from cupric oxide nano line,
Titanium dioxide nano thread, nanowires of gold, nano silver wire, copper nano-wire, titanium nano wire, Fe nanowire, Pt nanowires,
Any of titanium platinum alloy nano wire, iron oxide nano-wire, stannic oxide nano wire, silicon nanowires of metallic cover.
3. cell electrotransfection device according to claim 1, it is characterised in that the direction of growth of the nano wire is consistent.
4. cell electrotransfection device according to claim 1, it is characterised in that the nano wire is given birth to perpendicular to substrate surface
It is long.
5. cell electrotransfection device according to claim 1, it is characterised in that the diameter range of the nano wire is
30nm~5 μm, length range is 3 μm~300 μm.
6. cell electrotransfection device according to claim 1, it is characterised in that two electrodes are fixed on by electrode fixed mount
In electrotransfection container.
7. cell electrotransfection device according to claim 1, it is characterised in that the distance between two electrodes scope is 0.5
Mm~5mm.
8. cell electrotransfection device according to claim 1, it is characterised in that the electrotransfection container be shaped as tubulose,
Any of cylindrical type, cuboid, polygonal.
9. a kind of method that electrotransfection is carried out using the cell electrotransfection device as described in claim 1~8 any claim, including such as
Lower step：
(1) in electrotransfection container, cell to be transfected and material to be transfected are added；
(2) electrotransfection control circuit is opened, electrotransfection is carried out.
10. electrotransfection method according to claim 9, it is characterised in that methods described is the electric pulse of non-treatment diagnostic purpose
The transfection method of auxiliary.
11. electrotransfection method according to claim 9, it is characterised in that in step (1), the cell to be transfected is
Plant cell, zooblast or human cell.
12. electrotransfection method according to claim 9, it is characterised in that in step (1), the cell choosing to be transfected
From stem cell, DC cells, macrophage, lymphocyte.
13. electrotransfection method according to claim 9, it is characterised in that in step (1), the cell to be transfected
Density range is 5 × 105Individual/ml~5 × 107Individual/ml.
14. electrotransfection method according to claim 9, it is characterised in that in step (1), at the cell to be transfected
In static or flow regime.
15. electrotransfection method according to claim 9, it is characterised in that in step (1), when the cell to be transfected
During in flow regime, flowing velocity is 300L/ (hm2)~900L/ (hm2)。
16. electrotransfection method according to claim 9, it is characterised in that in step (1), the material to be transfected is selected from
DNA, siRNA, microRNA, albumen, polypeptide.
17. purposes of the cell electrotransfection device in cell electrotransfection instrument is prepared as described in claim 1~8 any claim.
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| CN111172034A (en) * | 2020-02-21 | 2020-05-19 | 中山大学 | Adjustable voltage mode cell perforation and membrane permeation system based on nanotube array sensor |
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| CN111808885A (en) * | 2019-04-12 | 2020-10-23 | 苏州壹达生物科技有限公司 | Electrotransfection device utilizing air bag pressurization |
| CN111808880A (en) * | 2020-08-10 | 2020-10-23 | 广州来恩生物医药有限公司 | Electrotransfection buffer solution and application thereof |
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| USD1016324S1 (en) | 2020-07-08 | 2024-02-27 | NanoCav, LLC | Biological cell processing chip |
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