CN106995783B - Cell electrotransfection device and its application - Google Patents
Cell electrotransfection device and its application Download PDFInfo
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- CN106995783B CN106995783B CN201610044870.8A CN201610044870A CN106995783B CN 106995783 B CN106995783 B CN 106995783B CN 201610044870 A CN201610044870 A CN 201610044870A CN 106995783 B CN106995783 B CN 106995783B
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Abstract
The invention belongs to cell transfection technique fields, and in particular to a kind of cell electrotransfection device and its application.The cell electrotransfection device, including at least an electrotransfection control circuit, two electrodes and an electrotransfection container, two electrodes are connect with the power supply in electrotransfection control circuit, one of electrode is as positive electrode, another electrode is as negative electrode, two electrodes are set in the electrotransfection container, and the surface of one or two of described two electrodes electrode has nanowire structure.Cell electrotransfection device of the invention can be used in the transfection of various kinds of cell, and cell survival rate is high, and transfection is good, has very high application value.
Description
Technical field
The invention belongs to cell transfection technique fields, and in particular to a kind of cell electrotransfection device and its application.
Background technique
Cell transfecting is that one kind of the importing cell such as exogenous DNA, RNA, albumen, polypeptide, small molecule compound is special
Gate technique can be applied to eukaryocyte, bacterium.With the continuous development of molecular biology and RESEARCH ON CELL-BIOLOGY, transfection is
Through becoming the conventional tool studied and control cytogene function.Research gene function, controlling gene expression, mutation analysis and
In the biological tests such as protein production, using more and more extensive.
Cellular immunity based on Chimeric antigen receptor (CAR) becomes the emerging form for the treatment of malignant tumour at present.CAR
Modification T cell is that the single-chain antibody and the activation motifs of T cell that will identify tumor associated antigen are combined as a whole, i.e., by antibody pair
The high-affinity of tumour antigen is combined with the killing mechanism of T lymphocyte, by gene transfer method transfecting T cells, makes it
Ability with specific recognition and killing tumor cell.
CAR-T immunotherapy is that T cell is isolated from blood samples of patients, after then carrying out genetic modification to it, makes T cell
Surface expression Chimeric antigen receptor.Patient's body is fed back to after laboratory expands these T cells, it is thin using these T
Born of the same parents carry out accurate immune attack to tumour cell.
Therefore, the immunocyte of CAR modification needs gene transfer method efficiently, safe.Clinically it is used to carry at present
The carrier of CAR is mainly derived from retrovirus and slow virus.Wherein retrovirus using more mature, by virus
The acceptor interaction of membrane glycoprotein and host cell surface and enter host cell, later reverse transcriptase starting synthetic DNA simultaneously
Random integration, being capable of stable transfection particular host cell into host genome.But this method can only transfect the cell of division stage,
And there are problems that some potential safety problems: first:, can be to autologous patient since there are radom insertions to integrate for transcription vector
Cell causes irreversible genetic manipulation, there is the potential risk for generating canceration；Second: CAR-T cell is usually with reversion at present
The periphery blood T lymphocyte of record or slow-virus transfection, screens in vitro and amplifies stable expression cell.When treatment complete or
How these transgenic T cells are disposed when side effect occurs in person as a future trouble problem；Third: viral vectors transgenosis skill
Art is operationally cumbersome, will lead to the unfavorable factor for leading to the problem of quality management and control.Safety problem turns as viral vectors
The inherent shortcoming of gene.
Non-virus carrier transgenic method common are lipofection and electrotransfection method.Lipofection is transfected
Efficiency is limited, and is not suitable for clinical application 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 make
Nucleic acid actively enters cell under electric field action.The commercialization electroporation apparatus being widely used at present, mostly uses parallel aluminum
Electrode needs the up to voltage of 200V or more and transfection reagent box to assist, can be generated under action of high voltage stronger fuel factor and
Electrochemical effect, metal electrode generates in electrochemical reaction metal cation and generates toxicity to cell, and cell is subject to
The factors such as the inhomogeneities of electric field action cause the death rate of cell higher, and transfection is inhomogenous.
The Gene Pulser MXcell electroporation output waveform of Bio-Rad company is exponential wave or square wave, most cells
Transfection voltage be 200-400V, and need using matched electroporation buffer.
The Neon electroporation output waveform of Life technology company is only square wave, output voltage 500-2500V, more
The transfection voltage of number cell is greater than 1000V, and needs to turn buffer using corresponding electricity according to the difference of cell type.
In recent years, nano material electrode is also applied to drug conveying intracellular.PEI modification silicon nano-array material by with
Carry out transfection mammalian cell, but rely solely on nano wire to the penetration of cell membrane, transfection efficiency is relatively low.
Nano material is used as to the electrode of electrotransfection, under the action of extra electric field power, higher transfection effect can be generated
Rate.For nano material electrode compared with ordinary electrode, special electrode surface microstructure can make local field strength increase by 3~4
A order of magnitude, so that applied voltage be made to substantially reduce, in addition its nanoscale can preferably interact with cell, Neng Gouke
Traditional electrotransfection technology high voltage and the low limitation of cell survival rate are taken, thus there is very big application value.
Retrieval discovery to the prior art, Xiexi et al. have delivered an entitled " Nanostraw on " ACSNANO "
Electroporation System for Highly Efficient Intracellular Delivery and
The article of Transfection ", this article is using the method for atomic layer deposition and reactive ion etching in a kind of the porous of commercialization
It is prepared for alumina nano tube in polycarbonate membrane, tube diameters 250nm, 1.5 μm of length, in voltage 6-20V, pulsewidth 20-
200 μ s, cell survival rate 90% under conditions of 200-2000 pulse, transfection efficiency reaches 70%.However, this method or main
Using nanotube to the mechanism of attached cell, the electrophoretic action of electric field is transfected.And the suspension cell of small volume is not
It can be attached to nanotube substrate surface, and can not be transfected with the device.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide one kind to be based on nano line electrode
Cell electrotransfection device and its application.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of cell electrotransfection device, includes at least electrotransfection control circuit, two
A electrode and an electrotransfection container, two electrodes are connect with the power supply in electrotransfection control circuit, and one of electrode is made
For positive electrode, another electrode is as negative electrode, and two electrodes are set in the electrotransfection container, in described two electrodes
The surface of one or two electrode has nanowire structure.
Preferably, the nano wire is selected from cupric oxide nano line, titanium dioxide nano thread, nanowires of gold, silver nanowires, copper
Nano wire, titanium nano wire, Fe nanowire, Pt nanowires, titanium platinum alloy nano wire, iron oxide nano-wire, stannic oxide nano wire,
Any one of silicon nanowires of metallic cover.
When the material of only one electrode in two electrodes is nano wire, the nano wire directly can respectively be received selected from above-mentioned
Any one of rice noodles.
When the material of two electrodes is nano wire, the nano-material of two electrodes can be identical, can not also phase
Together.
Preferably, the direction of growth of the nano wire is consistent.
Preferably, the nano wire is grown 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 receive
The diameter of rice noodles must be in 100nm or less.
Preferably, the diameter range of the nano wire are as follows: 30nm~5 μm.It is further preferred that the diameter range of the nano wire
Are as follows: 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 exists
Between 10 μm~100 μm.
It is further preferred that the nano wire is selected from CuO nano wire, the diameter range of the CuO nano wire 50nm~
Between 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 model of the titanium dioxide nano thread
It is trapped among 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 100nm~
Between 150nm, length range is between 10 μm~15 μm.
Preferably, two electrodes are fixed in electrotransfection container by electrode fixed frame.
Preferably, the distance between two electrodes range 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 used
The general source DG1022U of signal generator is as electrotransfection power supply.
Preferably, the shape of the electrotransfection container is any one of tubulose, cylindrical type, cuboid, polygonal.
Method in the prior art can be used and prepare nano wire in aforementioned electrotransfection device, such as: thermal oxidation method, template
Method, chemical vapour deposition technique.
The second aspect of the present invention provides a kind of method for carrying out electrotransfection using aforementioned electrotransfection device, including such as
Lower step:
(1) in electrotransfection container, cell to be transfected and substance to be transfected is added；
(2) electrotransfection control circuit is opened, electrotransfection is carried out.
Preferably, aforementioned electrotransfection device is used to carry out the method for electrotransfection as the electrotransfection side of non-treatment diagnostic purpose
Method.
Preferably, in step (1), the cell to be transfected is selected from suspension cell or attached cell.It is further preferred that institute
It states cell to be transfected and is selected from stem cell, DC cell, macrophage, lymphocyte.
Preferably, in step (1), the density range of the cell to be transfected is 5 × 105A/ml~5 × 107A/ml.
Preferably, in step (1), the cell to be transfected is in static or flow regime.It is further preferred that when described
When cell to be transfected is in flow regime, flowing velocity is 300L/ (hm2)~900L/ (hm2)。
Preferably, in step (1), the substance to be transfected is selected from DNA, siRNA, microRNA, albumen, polypeptide.
Preferably, in step (2), the electrotransfection control circuit uses signal generator.It is further preferred that the letter
Number generator selects general source DG1022U.
Preferably, third aspect present invention provides aforementioned cells electrotransfection device in preparing cell electrotransfection instrument
Purposes.
Compared with prior art, the invention has the following beneficial effects:
The present invention after extensive and in-depth study, constructs the cell electrotransfection device based on nano line electrode first.
Cell electrotransfection device of the invention, used transfection voltage are less than or equal to 10V, applicable cell type be include a variety of thin
Various adherent and suspension cell including born of the same parents system and primary cell.Also that is, under conventional low-voltage, effect can be realized and be up to 20
The electrotransfection of ten thousand RLU, and Transfected cells survival rate is above 60% or more.
In conclusion cell electrotransfection device manufacture craft of the invention is simple, low in cost, in use, entire transfection
Process is simple to operation, can be used in the transfection of various kinds of cell, and cell survival rate is high, transfection is good, has very high
Application value.
Detailed description of the invention
Fig. 1: copper mesh oxidation front and back outside drawing in the embodiment of the present invention.
Fig. 2: CuO nano wire SEM schemes in the embodiment of the present invention.
Fig. 3: electrotransfection electrode schematic diagram.
Fig. 4: electrotransfection electrode fixed frame schematic diagram.
Fig. 5: electrotransfection schematic device.
Fig. 6 A: the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode is for the suitable of Chinese hamster ovary celI
Suitable transfection conditions are as follows: voltage 8V, pulsewidth 30ms, period 1s, pulse number 20 times.
Fig. 6 B: using the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode and it is directed to CHO
The suitable transfection conditions of cell: voltage 8V, pulsewidth 30ms, period 1s pulse number 20 times, use Opti-MEM, Hepes respectively
Buffer (10mM HEPES, 272mM sucrose, pH7.3), sucrose phosphate buffer (10mM phosphate, 272mM sucrose,
PH7.3 electrotransfection) is carried out, illustrate, when electrotransfection Chinese hamster ovary celI, buffer uses Opti-MEM transfection preferable.
Fig. 7: the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode is directed to RAW264.7 cell
Suitable transfection conditions are as follows: voltage 8V, pulsewidth 10ms, period 1s, pulse number 8 times.
Fig. 8: the cell electrotransfection device constructed by embodiment 5 containing titanium dioxide nano line electrode is directed to Chinese hamster ovary celI
Suitable transfection conditions are as follows: voltage 8V, pulsewidth 30ms, period 1s, pulse number 15 times.
Fig. 9: the cell electrotransfection device constructed by embodiment 5 containing titanium dioxide nano line electrode is thin for Jurkat
The suitable transfection conditions of born of the same parents are as follows: voltage 10V, pulsewidth 10ms, period 1s, pulse number 14 times.
Figure 10: the cell electrotransfection device constructed by embodiment 6 containing nanowires of gold electrode is for the suitable of Chinese hamster ovary celI
Transfection conditions are as follows: voltage 8V, pulsewidth 30ms, period 1s, pulse number 20 times.
Figure 11: the cell electrotransfection device constructed by embodiment 6 containing nanowires of gold electrode is for Jurkat cell
It is suitable for transfection conditions are as follows: voltage 8V, pulsewidth 10ms, period 1s, pulse number 16 times.
Specific embodiment
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and 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
INMOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe,
CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；
METHODSIN 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 1 CuO nano line electrode of embodiment
The present embodiment prepares CuO nano line electrode using thermal oxidation method, specifically comprises the following steps:
Purity is greater 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 impregnate about 30min, to dissolve table
The oxide and impurity in face.Deionized water repeated flushing is used again, until washing lotion is neutral, finally, with being dried with nitrogen.It will be clear
Wash it is dry after copper mesh be put into quartz boat, be sent into ramped heating schedule furnace, in the atmospheric atmosphere of 0.03MPa, with 5 DEG C/
The heating rate of min increases temperature to 500 DEG C, then after 500 DEG C of heating 2h, cools to room temperature with the furnace.It after reaction can be with
See that copper mesh surface is covered by the oxide layer of one layer of black.Copper mesh oxidation front and back appearance is as shown in Figure 1.By copper mesh thermal oxide legal system
Standby obtained CuO nano wire, is observed in the secure execution mode (sem, as a result as shown in Fig. 2, the CuO nanowire growth direction it is consistent, perpendicular to
Copper-based growth, bottom end is thick, top is sharp.The diameter range of the CuO nano wire is between 50~150nm, and length is at 10~20 μm.
In addition, existing other technologies can also be used to prepare CuO nano wire, as long as prepared CuO nanometer line morphology, diameter and length
Degree meets the requirements.
The building of 2 cell electrotransfection device of embodiment
Using CuO nano wire prepared in embodiment 1 as electrode (as shown in Figure 3), cell electrotransfection device is constructed.
The electrotransfection device includes at least an electrotransfection control circuit, two CuO nano line electrodes and an electrotransfection and holds
Device.The prior art, such as signal generator (general source DG1022U) can be used in the electrotransfection control circuit, can generate it is accurate,
Stablize, the output signal of low distortion, includes five kinds of reference waveforms such as square wave, pulse, sine wave, and soft using any wave editor
Waveform required for part Ultrawave is exported, output voltage range 2mV~10V, 1 μ Hz~25MHz of frequency range；It can be according to electricity
Turn the parameters such as waveform required for the different flexible modulations of cell, voltage, pulsewidth, pulse number.Two CuO nano line electrodes with
Power supply connection in electrotransfection control circuit.Two CuO nano line electrodes can be set in electrotransfection container, and be fixed by electrode
Frame is (as shown in Figure 4) fixed.One of electrode can be used as anode, another can be used as cathode, after energization, can form electricity
, electrotransfection is carried out to the cell for being placed in electrotransfection container.
As shown in figure 5, the building process of electrotransfection device, may include step:
CuO nano line electrode 2 is placed on the platform of fixed frame bottom by the first step, and blend rubber circle compresses；Second
Step, CuO nano line electrode 1, which is placed on cell suspension container, (also that is, electrotransfection container, such as can be used the training of 24 holes of commercialization
Support plate) bottom；Fixed frame is inserted into cell suspension container, and electrode 1 is compressed by third step；The bottom of electrode fixed frame
Podium level is used to control the spacing of parallel pole 1 and electrode 2 in 1~2mm；4th step receives CuO nano line electrode 1 and CuO
Rice noodles electrode 2 is respectively adopted the conducting wire with crocodile clip and connect with signal generator (general source DG1022U), and passes through signal generator
Apply voltage；Turn operation step 5: cell suspension is added in cell suspension container and can carry out electricity.
3 cell electrotransfection device of embodiment transfects attached cell
Attached cell, this reality are transfected with the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode
Example is applied to illustrate by taking Chinese hamster ovary celI as an example.
Transfection method includes the following steps:
(1) on the day before electroporation (electrotransfection), with proper density passage cell, make cell before transfection in logarithmic growth
Phase, cell monolayer degrees of fusion about 70-80%；
(2) two CuO nano line electrodes, electrode fixed frame and washer are placed in 75% alcohol and are impregnated 2 hours, then existed
It can be used after irradiating 30min under ultraviolet lamp, CuO nano line electrode placed in the way of in embodiment 2 and electrode is fixed
Frame；
1ml DMEM culture medium (containing 10% serum, be free of antibiotic) is added in every hole in (3) 6 porocyte culture plates, is placed on
37 DEG C of CO2It is preheated in incubator；
(4) the logarithmic phase cell for taking out culture discards culture medium, after PBS washs cell, trypsin digestion cell 2min, to
After cell rounding, is terminated and digested with DMEM complete medium；
(5) after gently blowing down cell into single cell suspension, 1000rpm room temperature is centrifuged 5min；
(6) it discards supernatant completely, PBS is added, cell is resuspended, counted after blowing and beating uniformly, and according to cell density, taken certain
The re-suspension liquid of volume；
(7) 1000rpm room temperature is centrifuged 5min, discards supernatant completely, and Opti-MEM culture medium is added and is resuspended and blows and beats uniformly,
It is added and is mixed to Pignus pignoris grain pGL3, make final cell densities 5 × 106A/ml, the final concentration of 0.1mg/ml of plasmid；It is soft up and down
Piping and druming is uniform；
(8) 250 μ l cell suspensions are added into the cell culture plate well for assemble electrode；By the positive and negative anodes of signal generator
It is connected on two plate electrodes (electrode 2 and electrode 1) up and down, applies voltage；Electricity turns condition: voltage 8V, pulsewidth 50ms, frequency 1Hz,
Pulse number 10 times；And turn condition using other electricity as shown in FIG. 6, carry out electrotransfection；
(9) cell suspension after electric shock is added in the culture medium of preheating, up and down after piping and druming uniformly, puts back to 37 DEG C of CO2
It is normally cultivated in incubator；Electricity carries out cell survival rate detection after turning immediately；And after transfection for 24 hours, Luciferase table is carried out
Up to detection.
Cell survival rate detection method:
In (1) 96 orifice plate, every hole is previously added 90 μ l culture mediums；
(2) it after electricity turns, takes 10 μ l electricity to turn cell suspension, is added in 96 orifice plates；
(3) then, under the conditions of being protected from light, 10 μ l CCK8 reaction substrates are added in every hole；
After being incubated for 1-4h in (4) 37 DEG C of cell incubators, with the absorbance in microplate reader measurement each hole at 450nm wavelength
Value；
(5) cell survival rate (%)=[A (electricity turns)-A (blank)]/[A (non-electrical turns)-A (blank)] × 100
A (electricity turns): there is CCK8 solution, turn the absorbance in the cell suspension hole of processing through electricity；
A (non-electrical turns): having CCK8 solution, and not doing the cell suspension hole that electricity turn is handled, (use is identical with electric turn hole slow
Fliud flushing suspension cell) absorbance；
A (blank): there is CCK8 solution, electricity to turn buffer without the absorbance in the hole of cell.
Luciferase detection of expression method:
(1) after cell transfecting 24 hours, culture medium is discarded, PBS is washed one time；
(2) 5 × cell pyrolysis liquid (Promega) is diluted to 1 with pure water ×, and restore using preceding to room temperature；
(3) every hole is added 150 μ l1 × cell pyrolysis liquid and rocks, it is made sufficiently to cover cell, is put into -30 degree refrigerators
Freeze thawing 30min；
(4) cell is scraped from board bottom, and all liq is transferred in centrifuge tube, 12,000g centrifugations take supernatant；
(5) the above-mentioned lysate of 50 μ l and 20 μ l Luciferase measurement liquid are mixed；
(6) it is immediately placed in fluorescence illumination photometer and measures, waiting time 0.6s, time of measuring 10s.
Cell Transfection Conditions optimization:
The conditions such as voltage, pulsewidth, period, pulse number are optimized, as a result as shown in Figure 6A: constructed by embodiment 2
The cell electrotransfection device containing CuO nano line electrode be directed to Chinese hamster ovary celI suitable transfection conditions are as follows: voltage 8V, pulsewidth
30ms, period 1s, pulse number 20 times.
In addition, the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode is carried out for Chinese hamster ovary celI
Electrotransfection, under each transfection conditions as shown in Figure 6A, cell survival rate is above 60%.
Using the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode and for Chinese hamster ovary celI
It is suitable for transfection conditions: voltage 8V, pulsewidth 30ms, period 1s pulse number 20 times, uses Opti-MEM, Hepes buffer respectively
(10mM HEPES, 272mM sucrose, pH7.3), sucrose phosphate buffer (10mM phosphate, 272mM sucrose, pH7.3) carry out
Electrotransfection, as a result as shown in Figure 6B, when electrotransfection Chinese hamster ovary celI, buffer is preferable using Opti-MEM transfection, and up to 200,000
RLU。
4 cell electrotransfection device Transfection of macrophages of embodiment
With the cell electrotransfection device Transfection of macrophages constructed by embodiment 2 containing CuO nano line electrode, this reality
Example is applied to illustrate by taking RAW264.7 cell as an example.
Transfection procedure is as follows:
(1) on the day before electroporation, with proper density passage cell, make cell before transfection in logarithmic growth phase, cell
Single layer of confluent degree about 70-80%；
(2) electrode, fixed frame and washer are placed in 75% alcohol and are impregnated 2 hours, then irradiate 30min in the UV lamp
It can be used afterwards, electrode and fixed frame placed in the way of in embodiment 2；
(3) every hole addition 1ml RPMI-1640 culture medium (contains 10% serum, 20ng/ml GM-CSF is free of in 6 orifice plates
Antibiotic), it is placed in 37 DEG C of CO2 incubator and preheats.
(4) the logarithmic phase cell for taking out culture, discards culture medium, is blown and beaten cell from culture dish bottom with the PBS of pre-cooling
Get off.
(5) it will be counted after cell piping and druming uniformly, and according to cell density, take the cell suspension of certain volume.
(6) 1000rpm room temperature is centrifuged 5min, discards supernatant completely, and Opti-MEM culture medium is added and is resuspended and blows and beats uniformly,
It is added and is mixed to Pignus pignoris grain pGL3, make final cell densities 107A/ml, the final concentration of 0.1mg/ml of plasmid.Soft piping and druming up and down
Uniformly.
(7) into the cell culture plate well for assembling electrode, 250 μ l cell suspensions are added in every hole；By signal generator
Positive and negative anodes are connected on two plate electrodes (electrode 2 and electrode 1) up and down, apply voltage；Electricity turns condition: voltage 8V, pulsewidth 10ms, frequency
Rate 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 uniformly, puts back to 37 DEG C of CO2Culture
It is normally cultivated in case；Electricity carries out cell survival rate detection after turning immediately；And after transfection for 24 hours, Luciferase expression inspection is carried out
It surveys.
Cell survival rate detection method: cell survival rate detection method described in reference implementation example 3.
Luciferase detection of expression method: Luciferase detection of expression method described in reference implementation example 3.
Cell Transfection Conditions optimization:
The conditions such as voltage, pulsewidth, period, pulse number are optimized, as a result as shown in Figure 7: constructed by embodiment 2
Cell electrotransfection device containing CuO nano line electrode is directed to the suitable transfection conditions of RAW264.7 cell are as follows: voltage 8V, pulsewidth
10ms, period 1s, pulse number 8 times.
In addition, the cell electrotransfection device constructed by embodiment 2 containing CuO nano line electrode is directed to RAW264.7 cell
Electrotransfection is carried out, under each transfection conditions as shown in Figure 7, cell survival rate is above 60%.
The preparation of 5 titanium dioxide nano line electrode of embodiment, the building of cell electrotransfection device, cell transfecting
The present embodiment prepares titanium dioxide nano line electrode using thermal oxidation method, specifically referring to the preparation method of embodiment 1
Include the following steps:
The titanium net that will be cleaned up is placed in ramped heating schedule furnace, in the atmospheric atmosphere of 0.03MPa, with 5 DEG C/min
Heating rate increase temperature to 750 DEG C, then after 750 DEG C of heating 10h, cool to room temperature with the furnace.The titanium dioxide being prepared
For titanium nano wire perpendicular to substrate surface, the direction of growth is consistent, and for diameter range between 150~200nm, length is about 5~100 μ
m。
In addition, existing other technologies can also be used to prepare titanium dioxide nano thread, as long as prepared titanium dioxide
Nanometer line morphology, diameter and length meet the requirements.
Cell is constructed using titanium dioxide nano thread prepared by the present embodiment as electrode referring to the method for embodiment 2
Electrotransfection device.
The electrotransfection device, include at least an electrotransfection control circuit, two titanium dioxide nano line electrodes and
One electrotransfection container.The prior art, such as signal generator (general source DG1022U) can be used in the electrotransfection control circuit,
Accurate, stable, low distortion output signal can be generated, includes five kinds of reference waveforms such as sine wave, square wave, pulse, and can benefit
Waveform required for being exported with any wave software for editing Ultrawave, output voltage range 2mV~10V, 1 μ Hz of frequency range~
25MHz；The parameters such as waveform, voltage, pulsewidth, pulse number required for can turning the different flexible modulations of cell according to electricity.Two
Titanium dioxide nano line electrode is connect with the power supply in electrotransfection control circuit.Two titanium dioxide nano line electrodes can be set to electricity
It transfects in container, and is fixed by electrode fixed frame.One of electrode can be used as anode, another can be used as cathode, lead to
After electricity, electric field can be formed, electrotransfection is carried out to the cell for being placed in electrotransfection container.
It is adherent thin with the cell electrotransfection device transfection constructed by the present embodiment 5 containing titanium dioxide nano line electrode
Born of the same parents, the present embodiment illustrate by taking Chinese hamster ovary celI as an example.Specific transfection method, cell survival rate detection method and Luciferase
Detection of expression method is referring to embodiment 3.
The conditions such as voltage, pulsewidth, period, 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 directed to the suitable transfection conditions of Chinese hamster ovary celI are as follows: voltage 8V, pulsewidth
30ms, period 1s, pulse number 15 times.
In addition, the cell electrotransfection device constructed by embodiment 5 containing titanium dioxide nano line electrode is directed to Chinese hamster ovary celI
Electrotransfection is carried out, under each transfection conditions as shown in Figure 8, cell survival rate is above 60%.
It is thin with the cell electrotransfection device transfection lymph constructed by the present embodiment 5 containing titanium dioxide nano line electrode
Born of the same parents, the present embodiment illustrate by taking suspension cell Jurkat as an example.
Transfection procedure is as follows:
On the day before electroporation, with proper density passage cell, make cell before transfection in logarithmic growth phase.
(1) electrode, fixed frame and washer are placed in 75% alcohol and are impregnated 2 hours, then irradiate 30min in the UV lamp
It can be used afterwards；Electrode and fixed frame are placed by mode described in embodiment 2；
1ml RPMI-1640 culture medium (containing 10% serum, be free of antibiotic) is added in every hole in (2) 12 orifice plates, is placed on 37
DEG C CO2It is preheated in incubator；
(3) the logarithmic phase cell of culture is taken out, 1000rpm room temperature is centrifuged 5min, discards culture medium；
(4) PBS is added and cell is resuspended, counted after blowing and beating uniformly, and according to cell density, take the re-suspension liquid of certain volume；
(5) 1000rpm room temperature is centrifuged 5min, discards supernatant completely, and Opti-MEM culture medium is added and is resuspended and blows and beats uniformly,
It is added and is mixed to Pignus pignoris grain pGL3, make final cell densities 107A/ml, the final concentration of 0.1mg/ml of plasmid.Soft piping and druming up and down
Uniformly；
(6) 250 μ l cell suspensions are added in every hole；The positive and negative anodes of signal generator are connected on two plate electrodes up and down, are applied
Making alive.Electricity turns condition: voltage 8V, pulsewidth 10ms, frequency 1Hz, and pulse number 8 times；
(7) cell suspension after electric shock is added in the culture medium of preheating, after piping and druming uniformly, puts back to 37 DEG C of CO2Culture
It is normally cultivated in case；Electricity carries out cell survival rate detection after turning immediately；And after transfection for 24 hours, Luciferase expression inspection is carried out
It surveys.
Specific cell survival rate detection method and Luciferase detection of expression method are referring to embodiment 3.
The conditions such as voltage, pulsewidth, period, 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 directed to the suitable transfection conditions of Jurkat cell are as follows: voltage 10V,
Pulsewidth 10ms, period 1s, pulse number 14 times.
In addition, the cell electrotransfection device constructed by embodiment 5 containing titanium dioxide nano line electrode is directed to Jurkat
Cell carries out electrotransfection, and under each transfection conditions as shown in Figure 9, cell survival rate is above 60%.
Preparation, the building of cell electrotransfection device, cell transfecting of 6 nanowires of gold electrode of embodiment
The present embodiment prepares nanowires of gold electrode using template, specifically comprises the following steps:
Purity is greater than 99.9%, the goldleaf with a thickness of 10 μm is placed in 1molL-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,
Cathode is the goldleaf cleaned in advance and polycarbonate perforated membrane (aperture: 100 nanometers；Film thickness: 6 millimeters；Hole density
107cm2), polycarbonate perforated membrane is tightly covered on goldleaf surface, and electrolyte component is to contain 0.5M HClO in electrolytic cell4's
Chlorauric acid solution (w/w 1%), carries out electro-deposition under the constant potential of 0.18V.After deposition, template is immersed in 4 DEG C
CHCl32h dissolves template molecule in solution, i.e. acquisition nanowires of gold electrode.According to the aperture of polycarbonate membrane, diameter is obtained
The nanowires of gold electrode of 100~150nm.Sedimentation time 15min is controlled, nanowire length is about 10~15 μm.
In addition, existing other technologies can also be used to prepare nanowires of gold, as long as prepared nanowires of gold form, straight
Diameter and length meet the requirements.
Cell electrotransfection is constructed using nanowires of gold prepared by the present embodiment as electrode referring to the method for embodiment 2
Device.
The electrotransfection device includes at least an electrotransfection control circuit, two nanowires of gold electrodes and an electricity
Transfect container.The prior art, such as signal generator (general source DG1022U) can be used in the electrotransfection control circuit, can produce
Production of sperm is true, stablizes, the output signal of low distortion, includes five kinds of reference waveforms such as sine wave, square wave, pulse, and using any
Waveform required for wave software for editing Ultrawave is exported, output voltage range 2mV~10V, 1 μ Hz~25MHz of frequency range；
The parameters such as waveform, voltage, pulsewidth, pulse number required for can turning the different flexible modulations of cell according to electricity.Two gold nanos
Line electrode is connect with the power supply in electrotransfection control circuit.Two nanowires of gold electrodes can be set in electrotransfection container, and be passed through
Electrode fixed frame is fixed.One of electrode can be used as anode, another can be used as cathode, after energization, can form electric field,
Electrotransfection is carried out to the cell for being placed in electrotransfection container.
Attached cell is transfected with the cell electrotransfection device constructed by the present embodiment 6 containing nanowires of gold electrode, this
Embodiment illustrates by taking Chinese hamster ovary celI as an example.Specific transfection method, cell survival rate detection method and Luciferase expression inspection
Survey method is referring to embodiment 3.The conditions such as voltage, pulsewidth, period, pulse number are optimized, the results are shown in Figure 10: being implemented
Cell electrotransfection device constructed by example 6 containing nanowires of gold electrode is directed to the suitable transfection conditions of Chinese hamster ovary celI are as follows: voltage
8V, pulsewidth 30ms, period 1s, pulse number 20 times.
In addition, the cell electrotransfection device constructed by embodiment 6 containing nanowires of gold electrode carries out electricity for Chinese hamster ovary celI
Transfection, under each transfection conditions as shown in Figure 10, cell survival rate is above 60%.
Lymphocyte is transfected with the cell electrotransfection device constructed by the present embodiment 6 containing nanowires of gold electrode, this
Embodiment illustrates by taking suspension cell Jurkat as an example.
The conditions such as voltage, pulsewidth, period, 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 directed to Jurkat cell suitable transfection conditions are as follows: voltage 8V, pulsewidth
10ms, period 1s, pulse number 16 times.
In addition, cell electrotransfection device constructed by embodiment 6 containing nanowires of gold electrode for Jurkat cell into
Row electrotransfection, under each transfection conditions as shown in figure 11, cell survival rate is above 60%.
The present inventor also uses silver nanowires, copper nano-wire, titanium nano wire, Fe nanowire, Pt nanowires, titanium platinum
Alloy nano-wire, iron oxide nano-wire, stannic oxide nano wire, metallic cover cell electrotransfection of the silicon nanowires as electrode
Device, the diameter range of each nano wire are as follows: 30nm~5 μm；Length range is between 3 μm~300 μm；It is preferred that each nano wire is straight
Diameter range are as follows: 50nm~1 μm；Length range is between 10 μm~100 μm.Through testing, electricity is carried out for various attached cells and is turned
Dye can obtain the cell survival rate and transfection similar with aforementioned three kinds of electrotransfection devices.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (8)
1. a kind of cell electrotransfection device includes at least an electrotransfection control circuit, two electrodes and an electrotransfection and holds
Device, two electrodes are connect with the power supply in electrotransfection control circuit, and one of electrode is made as positive electrode, another electrode
For negative electrode, two electrodes are set in the electrotransfection container, and the surface of one or two of described two electrodes electrode has
There is nanowire structure, the cell electrotransfection device includes the following steps: that (1) holds in electrotransfection when being used for cell electrotransfection
In device, cell to be transfected and substance to be transfected is added；(2) electrotransfection control circuit is opened, electrotransfection is carried out.
2. cell electrotransfection device according to claim 1, which is characterized in that the nano wire is selected from cupric oxide nano
Line, titanium dioxide nano thread, nanowires of gold, silver nanowires, copper nano-wire, titanium nano wire, Fe nanowire, Pt nanowires, titanium platinum
Any one of alloy nano-wire, iron oxide nano-wire, stannic oxide nano wire, silicon nanowires of metallic cover.
3. cell electrotransfection device according to claim 1, which is characterized in that the direction of growth of the nano wire is consistent.
4. cell electrotransfection device according to claim 1, which is characterized in that the nano wire is raw perpendicular to substrate surface
It is long.
5. cell electrotransfection device according to claim 1, which is characterized 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, which is characterized in that two electrodes are fixed by electrode fixed frame
In in electrotransfection container.
7. cell electrotransfection device according to claim 1, which is characterized in that the distance between two electrodes range is
0.5mm~5mm.
8. cell electrotransfection device according to claim 1, which is characterized in that the shape of the electrotransfection container is pipe
Any one of shape, cylindrical type, cuboid, polygonal.
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| DE112019003498T5 (en) | 2018-07-09 | 2021-04-08 | NanoCav, LLC | MICROFLOW ELECTROPORATION DEVICES AND METHODS OF CELL TRANSFECTION |
| 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 |
| 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 |
| CN111690685A (en) * | 2020-06-29 | 2020-09-22 | 深圳赛桥生物创新技术有限公司 | Method for improving transfection efficiency of electroporation cell |
| USD1016324S1 (en) | 2020-07-08 | 2024-02-27 | NanoCav, LLC | Biological cell processing chip |
| 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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