CN113832192A - Device and method for introducing exogenous substance into cells - Google Patents
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Abstract
The present invention provides a device and method for introducing exogenous material into cells. In a first aspect, the invention provides an apparatus for introducing exogenous material into a cell, comprising a reaction module and a control module; the reaction module comprises a first storage unit, a second storage unit and electrodes, wherein the first storage unit comprises a substrate provided with a plurality of through holes, the substrate is used for placing cells, the second storage unit is communicated with the through holes and used for placing the exogenous substances, and the electrodes are respectively connected with the first storage unit and the second storage unit; the control module is connected with the electrode and used for controlling the electrode to generate a pulse signal. The device provided by the invention is beneficial to improving the survival rate of cells and the introduction efficiency of exogenous substances.
Description
Technical Field
The invention relates to the technical field of electroporation, in particular to a device and a method for introducing exogenous substances into cells.
Background
Transfection is the process by which eukaryotic cells acquire a new phenotype by the active or passive introduction of exogenous material under certain conditions. With the development of genetic engineering technology, how to introduce exogenous substances into cells with high efficiency becomes a new research hotspot. The transfection methods commonly used include chemical means including calcium phosphate precipitation, liposome (Lipofectamine 2000, etc.), cationic polymer (PEI, etc.), and chemical means having low transfection efficiency for cells with weak phagocytosis (e.g., primary cells, suspension cells, etc.) and cells with strong lysosomal capacity (e.g., macrophages, etc.), and thus methods for viral infection are required for such refractory cells. The principle of the virus infection method is to change genome, which has higher risk, and the application of virus infection is limited to a certain extent due to long selection and preparation period of virus, complex operation, high cost and limitation on the size of the inserted fragment. The electroporation method is a physical means, and an electric field is applied to form a potential difference on two sides of a cell membrane to disturb the arrangement of phospholipid bilayers and generate a technology for communicating membrane pores inside and outside the cell, so that exogenous substances, such as plasmids, RNA, proteins or polysaccharides and the like, have a certain probability of entering the cell within the duration time of the membrane pores. Electroporation transfection can effectively transfect cells which are difficult to transform, has no limit on the size of plasmids, is transient transfection, has low probability of being embedded into genomes, and is often used as a supplementary means of chemical and viral methods, so the electroporation technology has wide application in the fields of gene editing, embryo transformation, cell mechanism research, protein production, cell treatment and the like.
At present, common electroporation devices comprise a Gene Pulser Xcell of Bio-Rad, a Neon system of Thermo Fisher, a Nuclear effector of Lonza and the like, wherein the Gene Pulser Xcell adopts a cuvette type electric rotating cup, and the death rate of cells is reduced by adopting an electric rotating buffer solution and outputting simple square waves or exponential attenuation waves; the Neon system changes the electric rotating cup into the electric rotating suction head, so that the distance between electrodes is reduced, the electric field is more uniform, and the survival rate of cells is improved; the Nucleofector adds a reagent for promoting the plasmid to enter the cell nucleus, namely a nuclear transfer reagent, into the electrotransformation buffer solution so as to improve the transfection efficiency. However, the existing electroporation devices all have the problems of strong transfection randomness, high cell death rate, high experiment cost and the like, and how to improve the electroporation device to solve the problems is receiving more and more attention.
Disclosure of Invention
The invention provides a device for introducing exogenous substances into cells, which is used for solving the problems of strong transfection randomness, high cell death rate, high experiment cost and the like of a conventional electroporation device.
The invention also provides a method for introducing exogenous substances into cells, and the device can be used for transfecting the cells, so that the problem of high transfection randomness can be effectively solved, and the transfection success rate and the cell survival rate of the cells are improved.
In a first aspect, the invention provides an apparatus for introducing exogenous material into a cell, comprising a reaction module and a control module;
the reaction module comprises a first storage unit, a second storage unit and electrodes, wherein the first storage unit comprises a substrate provided with a plurality of through holes, the substrate is used for placing cells, the second storage unit is communicated with the through holes and used for placing the exogenous substances, and the electrodes are respectively connected with the first storage unit and the second storage unit;
the control module is connected with the electrode and used for controlling the electrode to generate a pulse signal.
The above device, the through holes have a pore diameter of 50nm-8 μm and a height of 5 μm-50 μm, and the density of the through holes on the substrate is 1 × 104Per cm2-4*108Per cm2。
In the above device, the substrate includes a silicon-containing inorganic substance or a polymer, wherein the silicon-containing inorganic substance is selected from one or more of elemental silicon, silicon oxide and silicon nitride, and the polymer is selected from one or more of polycarbonate, polyethylene terephthalate and polyimide.
In the above device, the electrode includes a positive electrode connected to the first storage unit and a negative electrode connected to the second storage unit.
According to the device, the control module comprises a microcontroller unit, and a voltage generation unit, a current detection unit and a pulse switch unit which are connected with the microcontroller unit;
the voltage generation unit is used for receiving signals of the microcontroller unit and sending voltage, the voltage generation unit is connected with the current detection unit, the current detection unit is connected with the pulse switch unit, the current detection unit is used for detecting current of the reaction module and feeding back the current to the microcontroller unit, and the pulse switch unit is connected with the electrode and used for receiving signals of the microcontroller unit and controlling the switch of the pulse switch unit.
As with the device, the device comprises a power supply module which is used for providing power supply for the control module.
According to the device, the device comprises a touch display module, and the touch display module is connected with the control module and used for achieving man-machine interaction operation.
According to the device, the device comprises a sample rack, the sample rack comprises a side wall formed by enclosing, a first bulge and a second bulge are arranged on the side wall, the first bulge is used for bearing the first storage unit, and the second bulge is used for bearing the second storage unit.
In a second aspect, the present invention provides a method for introducing exogenous material into cells, using any of the above devices, comprising the steps of:
placing the cells in the logarithmic growth phase on the surface of the substrate, placing the solution containing the exogenous substances into the second storage unit, and immersing part of the substrate into the solution containing the exogenous substances, wherein the cells are not in contact with the solution containing the exogenous substances, so as to obtain a reaction system;
and electrifying the reaction system to enable the exogenous substances to enter the cells through the through holes under a pulse signal.
As above, the pulse signal includes a first pulse signal, a second pulse signal and a third pulse signal, wherein:
generating a first pulse signal by a control module, wherein a first voltage U of the first pulse signal11-3V, the width of the first pulse signal is 5-20ms, and the cell is obtained at a first voltage U1First current I of1And a first electricityResistance R1；
According to the first current I1And determining a first state from the number of cells, and determining a threshold value RI and a step voltage U from the first statestep；
Generating a second pulse signal by the control module, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2Greater than a first voltage U1By step voltage UstepGradually increasing, the width of the second pulse signal is 5-20ms, and the interval of the second pulse signal is 5-20 ms;
when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3Generating a third pulse signal when the change rate delta RI of the resistance of the cell along with the current is in a decreasing trend and is less than or equal to a threshold value RI, wherein the voltage of the third pulse signal is a third voltage U3The width of the third pulse signal is 10-50ms, the interval of the third pulse signal is 10-50ms, the number of the third pulse signals is 1-20, and the electrifying process is completed.
The implementation of the invention has at least the following advantages:
1. according to the device provided by the invention, the substrate with the through holes is arranged, so that the directional introduction of the exogenous substance is realized, the randomness of a common electroporation method is eliminated, and the introduction efficiency of the exogenous substance is improved; meanwhile, by applying a pulse signal, electroporation can be completed under a low voltage of 1-50V, the safety of electroporation is improved, and the survival rate of cells is improved.
2. The method provided by the invention has the advantages that the device is used for electroporation, the survival rate of cells and the introduction efficiency of exogenous substances are favorably improved, in addition, the design of separating the exogenous substances from the cells is adopted, the interference of additional factors on the cells is avoided, the purity of the system is improved, the operation and transfection process are simplified, and the experiment cost is reduced.
3. The method provided by the invention accurately controls the potential difference near the cell layer through a feedback and optimization algorithm, further protects the cells from being damaged by excessive current, and greatly improves the survival rate of the cells from 25% to over 90%.
4. The method provided by the invention can realize in-situ transfection, does not need to resuspend cells, greatly simplifies the operation process, and simultaneously makes the transfection of cells which can not be resuspended, such as primary neuron cells, possible.
5. The method provided by the invention does not need other auxiliary reagents, greatly reduces the experimental cost and is less than 1/10 in the prior art.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of an overall structure of an apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic structural view of a pre-culture apparatus according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a pulse signal according to an embodiment of the present invention;
FIG. 4a is a green fluorescence photograph of human embryonic stem cells under 488nm laser after 24 hours of the cells are introduced into EGFP plasmid;
FIG. 4b is a photograph of the cells provided in FIG. 4a in bright field;
FIG. 5a is a green fluorescence photograph of primary human PBMC cells under 488nm laser after being introduced with FAM-labeled siRNA fragment for 5 minutes;
FIG. 5b is a photograph of the cells provided in FIG. 5a in bright field;
FIG. 6a is a green fluorescence photograph of a HeLa cell provided by another embodiment of the present invention under 488nm laser after introducing Alexa Fluor 488 labeled H4B4 monoclonal antibody for 5 minutes;
FIG. 6b is a photograph of the cells provided in FIG. 6a in the bright field.
Description of reference numerals:
1: a reaction module;
1-1: a first storage unit;
1-1-1: a substrate;
1-1-2: a through hole;
1-2: a second storage unit;
1-3: a positive electrode;
1-4: a negative electrode;
2: a control module;
2-1: a microcontroller unit;
2-2: a voltage generating unit;
2-3: a current detection unit;
2-4: a pulse switching unit;
3: a power supply module;
4: a touch display module;
5: a sample holder;
5-1: a first protrusion;
5-2: a second protrusion;
6: a cell;
7: an exogenous substance;
8: a pre-incubator;
9: a pre-incubator lid;
10: a first pulse signal;
11: a second pulse signal;
12: and a third pulse signal.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In a first aspect of the present invention, a device for introducing exogenous substances into cells is provided, fig. 1 is a schematic diagram of an overall structure of the device provided in an embodiment of the present invention, as shown in fig. 1, the device includes a reaction module 1, a control module 2, a power module 3, a touch display module 4, and a sample holder 5, specifically:
the reaction module 1 is used for carrying out an electroporation experiment and comprises a first storage unit 1-1, a second storage unit 1-2, a positive electrode 1-3 and a negative electrode 1-4, wherein the first storage unit 1-1 is positioned above the second storage unit 1-2 and comprises a substrate 1-1-1 and a through hole 1-1-2 arranged on the substrate 1-1-1, the substrate 1-1-1 is used for placing a cell 6, the second storage unit 1-2 is used for placing an exogenous substance 7, the second storage unit 1-2 is communicated with the through hole 1-1-2, the substance exchange can be realized, namely, the exogenous substance 7 stored in the second storage unit 1-2 can enter the cell 6 through the through hole 1-1-2, the electroporation process is completed.
Because the substrate is used for placing cells, in order to improve the survival rate of the cells in the electroporation process, the surface of the substrate needs to be sterilized, and a material suitable for adherent culture of the cells is selected.
In one possible embodiment, the substrate includes a silicon-containing inorganic substance or a polymer, wherein the silicon-containing inorganic substance is selected from one or more of elemental silicon, silicon oxide and silicon nitride, and the polymer is selected from one or more of polycarbonate, polyethylene terephthalate and polyimide.
The through hole 1-1-2 is a through hole communicating the upper surface and the lower surface of the substrate 1-1-1, and a person skilled in the art can prepare the through hole by an etching process in the technology of preparing the substrate, and further, the through hole can be obtained by a deep silicon etching or track etching process because the through hole has a high aspect ratio (such as a nano-scale aperture and a micron-scale depth) and a good uniformity.
Since the size and density of the through-holes greatly affect the electroporation result, in order to further improve the introduction efficiency, the diameter of the through-holes is 50nm to 8 μm,the height is 5-50 μm, and the density of the through holes on the substrate is 1-104Per cm2-4*108Per cm2。
Further, the through holes have a pore diameter of 400nm-1 μm and a density of 2 × 10 on the substrate6Per cm2-1*108Per cm2。
The electrodes are connected with the first storage unit 1-1 and the second storage unit 1-2, wherein the electrodes comprise a positive electrode 1-3 and a negative electrode 1-4, it is understood that the direction of the electric field formed by the positive electrode and the negative electrode is related to the electrical property of the exogenous substance, since the exogenous substance is negatively charged in most cases, the positive electrode 1-3 is connected with the first storage unit 1-1, the negative electrode 1-4 is connected with the second storage unit 1-2, if the exogenous substance is positively charged, the electrode position needs to be reversed, that is, the positive electrode 1-3 is connected with the second storage unit 1-2, and the negative electrode 1-4 is connected with the first storage unit 1-1.
The materials of the positive and negative electrodes are conventional in the art, for example, the positive and negative electrodes are independently selected from one or more of aluminum, silver plating, conductive plastic, copper, stainless steel, gold plating, platinum gold, graphite.
The control module 2 is connected with the electrode and used for controlling the electrode to generate a pulse signal, and the control module 2 comprises a microcontroller unit 2-1, a voltage generation unit 2-2, a current detection unit 2-3 and a pulse switch unit 2-4 which are connected with the microcontroller unit 2-1;
the voltage generating unit 2-2 is used for receiving signals of the microcontroller unit 2-1 and sending out voltage, the voltage generating unit 2-1 is connected with the current detecting unit 2-3, the current detecting unit 2-3 is connected with the pulse switch unit 2-4, the current detecting unit 2-3 is used for detecting current of the reaction module 1 and feeding back the current to the microcontroller unit 2-1, and the pulse switch unit 2-4 is connected with an electrode and used for receiving signals of the microcontroller unit 2-1 and controlling the on-off of the pulse signals.
It can be understood that the pulse generation mode needs to be subjected to waveform generation, amplification and output, and after the pulse signal is amplified by the voltage generation unit, the electroporation can be completed at a low voltage of 1-50V, so that the safety of the electroporation is improved, and the survival rate of cells is improved.
In addition, because there is a pause between the pulse signals, the present embodiment uses the same line (i.e., voltage generation unit-current detection unit-pulse switch unit) to complete the process of reading-feedback-output, and the current value detected by the current detection unit is closer to the real situation.
The power module 3 is connected to the control module 2 for providing power to the control module 2, and can be set by a person skilled in the art according to conventional means.
The touch display module 4 is connected to the control module 2 for implementing human-computer interaction, for example, the control module 2 feeds back the power-on process to the touch display module 4 and receives the instruction of the touch display module 4 for operation.
The sample rack 5 is connected with the reaction module 1 and used for supporting a first storage unit 1-1 and a second storage unit 1-2, specifically, the sample rack 5 comprises a side wall formed by enclosing, the side wall is provided with a first protrusion 5-1 and a second protrusion 5-2, the first protrusion 5-2 is used for bearing the first storage unit 1-1, and the second protrusion 5-2 is used for bearing the second storage unit 5-3.
The device provided by the invention has the following operation process: the cell is placed on the surface of the substrate, the exogenous substance is placed in the second storage unit, the touch display module controls the start of electroporation, and in the electroporation process, the control module controls the electrodes to send out pulse signals, so that the exogenous substance enters the cell through the through holes.
In a second aspect, the present invention provides a method for introducing exogenous material into cells, using any of the above devices, comprising the steps of:
placing the cells in the logarithmic growth phase on the surface of the substrate, placing the solution containing the exogenous substances into the second storage unit, and immersing part of the substrate into the solution containing the exogenous substances, wherein the cells are not in contact with the solution containing the exogenous substances, so as to obtain a reaction system;
and electrifying the reaction system to enable the exogenous substances to enter the cells through the through holes under a pulse signal.
The present invention provides a method for introducing exogenous material into cells, wherein the cells used in the method can be resuspended cells commonly used in the art, or cells that cannot be resuspended, such as primary neuronal cells, and the exogenous material can be a material to be introduced into cells, such as plasmids, and the method for introducing exogenous material is not particularly limited, and the following method is described in detail:
step 1, placing the cells in the logarithmic growth phase on the surface of the substrate, placing the solution containing the exogenous substances into the second storage unit, and immersing part of the substrate into the solution containing the exogenous substances, wherein the cells are not in contact with the solution containing the exogenous substances, so as to obtain a reaction system:
firstly, culturing cells to a logarithmic growth phase, wherein the culturing process of the cells can be performed according to the conventional technical means in the field, for example, the cells are cultured by using a conventional culture dish or a multi-well plate, or the cells can be directly placed on the surface of the substrate in fig. 1 and placed in a cell culture box for culturing, fig. 2 is a schematic structural diagram of the cell pre-culture device provided by one embodiment of the invention, as shown in fig. 2, the device comprises a substrate 1-1-1, a pre-culture box 8 and a pre-culture box cover 9, the substrate 1-1-1 is placed inside the pre-culture box 8, and the pre-culture box cover 9 is covered for culturing; after a typical 24 hour incubation period, the cells enter the logarithmic growth phase, the substrate is removed and placed in the apparatus shown in FIG. 1 in preparation for electroporation transfection.
Next, the exogenous material is formulated into a solution having a concentration of 10. mu.g/mL to 80. mu.g/mL using sterile water and placed in a second storage unit.
Then, the substrate containing the cells in the logarithmic growth phase is immersed in the solution containing the exogenous substance, the cells are ensured not to be contacted with the solution containing the exogenous substance, no bubbles need to be needed in the placing process, otherwise, the current can be blocked at the position where the bubbles appear, so that part of the cells can not be subjected to electroporation, in addition, when the generated bubbles are large enough, the judgment of the subsequent current can be influenced, and the cell transfection efficiency is reduced.
Finally, the positive electrode and the negative electrode are connected with the reaction system, specifically, the positive electrode is inserted into the first storage unit, so that the front end of the upper electrode is immersed into the cell culture solution, and the negative electrode is placed below the second storage unit.
and then the control module can be opened to electrify the reaction system, exogenous substances positioned below can be directionally introduced into the cells along the direction of the through holes due to the through holes arranged on the surface of the substrate, and in order to ensure the survival rate of the cells, the invention uses pulse signals to carry out electroporation.
In order to further protect the cells from being damaged by excessive current, the invention precisely controls the potential difference of the cell membrane surface through algorithm optimization, specifically, the pulse signals comprise a first pulse signal, a second pulse signal and a third pulse signal, wherein:
generating a first pulse signal by a control module, wherein a first voltage U of the first pulse signal11-3V, the width of the first pulse signal is 5-20ms, and the first voltage U of the first system is obtained1First current I of1And a first resistor R1；
According to the first current I1And determining a first state from the number of cells, and determining a threshold value RI and a step voltage U from the first statestep；
Generating a second pulse signal by the control module, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2Greater than a first voltage U1By step voltage UstepGradually increasing, the width of the second pulse signal is 5-20ms, and the interval of the second pulse signal is 5-20 ms;
when the second electricityPress U2By step voltage UstepGradually increases to a third voltage U3And generating a third pulse signal when the change rate delta RI of the resistance of the cell along with the current is in a decreasing trend and is less than or equal to a threshold value RI, wherein the voltage of the third pulse signal is a third voltage U3The width of the third pulse signal is 10-50ms, the interval of the third pulse signal is 10-50ms, the number of the third pulse signals is 1-20, and the electrifying process is completed.
Fig. 3 is a schematic diagram of a pulse signal according to an embodiment of the present invention, as shown in fig. 3, the control module firstly forms a loop with the positive electrode and the negative electrode, outputs a first pulse signal 10 to the reaction system, and determines a first state according to a feedback current value and the number of cells, so as to determine a suitable pulse signal for a subsequent electroporation process, specifically, the first pulse signal 10 is a square wave, and a first voltage U of the first pulse signal is a square wave11-3V, the width of the first pulse signal is 5-20ms, and the obtained cell is at a first voltage U1First current I of1And a first resistor R1According to the first current I1And cell number determining the first state, as shown in table 1:
TABLE 1 first Current control Table (Current Unit: mA)
In table 1, the small, medium and large samples are determined according to the culture area of the cells on the substrate surface, and different cell resistances are caused based on different culture areas, and similarly, the difference in cell growth density in the same culture area causes the difference in resistance. In the specific implementation, the size and density of the sample are determined according to the conventional techniques for classifying the cells to be transfected, which is not limited by the present invention.
Then, a step voltage U is determined according to the first statestepAnd a threshold value RI, as shown in tables 2-3:
TABLE 2 second pulse signal parameter table (voltage unit: V)
TABLE 3 threshold RI Table (unit: Ω A)-1)
| StH1 | StH2 | StH3 | StM1 | StM2 | StM3 | StL1 | StL2 | StL3 | |
| RI | 32 | 27 | 15 | 35 | 30 | 18 | 40 | 35 | 22 |
It should be noted that the second voltages listed in table 2 are all 4V, which is determined according to the first voltage 1-3V, and in the actual electroporation process, a voltage greater than 3V can be selected, which is not limited to the 4V listed in table 2, and furthermore, if the first voltage is 1V, the second voltage can be 2V, which is only greater than the first voltage.
Then, a second pulse signal is generated by the control module, the interval between the first pulse signal and the second pulse signal is 100-2Is an initial voltage, the second voltage U2Greater than a first voltage U1By step voltage UstepGradually increasing, the width of the second pulse signal is 5-20ms, and the interval of the second pulse signal is 5-20 ms.
Finally, calculating the change rate delta RI of the resistance of the cell along with the current by the incremental current value fed back by the second pulse signal, and finding out a proper third pulse signal;
specifically, when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3When the Δ RI is decreasing and less than or equal to the threshold RI, triggering a third pulse signal, the voltage of which is a third voltage U3The width of the third pulse signal is 10-50ms, the interval of the third pulse signal is 10-50ms, the number of the third pulse signals is 1-20, and the electrifying process is completed.
The algorithm of the change rate Delta RI of the resistance along with the current is as follows:
firstly, calculating the resistance R of the sample under two adjacent pulses in the second pulse signal:
R2-1＝U2-1/I2-1，R2-2＝(U2-1+Ustep)/I2-2
U2-1、I2-1for voltage and current values under a single pulse, I2-2Is the current value at another single pulse;
secondly, calculating the change rate Delta RI of the resistance of the cell along with the current:
ΔRI＝(R2-1–R2-2)/(I2-2–I2-1)
and thirdly, the Delta RI tends to increase and then decrease, when the Delta RI attenuates to a threshold RI, a third pulse signal is generated, the third pulse signal is a square wave, the size of the third pulse signal is the voltage value of the last second pulse signal, the width of the third pulse signal is 10-50ms, the interval of the third pulse signal is 10-50ms, the number of the third pulse signals is 1-20, and the electroporation transfection is completed.
And finally, removing the electrode, putting the cells back to the original place for culturing, and observing fluorescence in the cells to judge the introduction/transfection efficiency of the exogenous substance.
The following detailed description is given in conjunction with specific examples:
example 1
The embodiment provides a method for introducing an EGFP plasmid into human embryonic stem cells, which specifically comprises the following steps:
culturing human embryonic stem cells to logarithmic growth phase by using a device shown in figure 2, and preparing an exogenous substance EGFP plasmid into a solution with the concentration of 40 mu g/mL by using sterile water;
cells in the log phase of growth were placed in the apparatus shown in fig. 1, 100 μ L of the EGFP plasmid-containing solution was placed in the second storage unit, and the substrate on which the human embryonic stem cells were placed was immersed in the EGFP plasmid-containing solution, and the human embryonic stem cells were not in contact with the EGFP plasmid-containing solution.
Controlling the touch display unit to generate a first pulse signal via the pulse switch unit, wherein the first voltage U is higher than the first voltage1Is 3V, the first pulse width is 10ms, and a first current I is obtained119.2 mA;
according to a first current I1Sample size and cell density were determined to be in the first state StH3 and a threshold resistance current change rate of 15 Ω A was determined according to tables 2-3-1Step voltage UstepIs 0.7V;
the touch display unit controls the pulse switch unit to generate a second pulse signal, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2The voltage is gradually increased by 0.7V at 4V, the pulse width is 5ms, and the pulse interval is 5 ms;
when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3And when the resistance change rate of the first system is less than or equal to the resistance threshold value, a third voltage U is applied3The touch display unit controls the pulse switch unit to generate a second pulse signal, a third voltage U39.6V, the third pulse width is 10ms, the pulse interval is 10ms, the number of pulses is 10, the electroporation transfection is completed, the human embryonic stem cells are put back into the cell culture solution for culture, and the fluorescence in the cells is observed to judge the introduction/transfection efficiency.
Fig. 4a is a photograph of green fluorescence of the human embryonic stem cell under 488nm laser after 24 hours of the introduction of the EGFP plasmid, fig. 4b is a photograph of the cell under bright field, as shown in fig. 4a-4b, most of the cells in fig. 4a have green fluorescence, which indicates that the EGFP plasmid is introduced into the cells, the transfection efficiency is high (more than 70%), and the cell state is good after electroporation.
Example 2
This example provides a method for introducing FAM-labeled siRNA fragments into primary PBMC cells, specifically comprising:
culturing primary PBMC cells to logarithmic growth phase by using the device shown in FIG. 2, and preparing siRNA fragments marked by exogenous FAM into a solution with the concentration of 100nM by using sterile water;
cells in the log phase of growth were placed in the apparatus shown in fig. 1 and temporarily attached to the substrate with a bio-gel (which automatically fails after 8 hours). Placing 100 μ L of the solution containing FAM-labeled siRNA fragments in a second storage unit, immersing the substrate with the primary PBMC cells placed therein in the solution containing FAM-labeled siRNA fragments, and the primary PBMC cells are not contacted with the solution containing FAM-labeled siRNA fragments.
Controlling the touch display unit to generate a first pulse signal via the pulse switch unit, wherein the first voltage U is higher than the first voltage1Is 3V, the first pulse width is 10ms, and a first current I is obtained115.6 mA;
according to a first current I1Cell size and cell density the first state was determined to be StH2 and the resistance threshold was determined to be 27 Ω A according to tables 2-3-1Step voltage UstepIs 1V;
the touch display unit controls the pulse switch unit to generate a second pulse signal, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2At 4V, with a step voltage UstepGradually increasing, the pulse width is 5ms, and the pulse interval is 5 ms;
when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3And when the resistance change rate of the first system is equal to the resistance threshold value, a third voltage U is applied3The touch display unit controls the pulse switch unit to generate a second pulse signal, a third voltage U310V, the third pulse width is 10ms, the pulse interval is 10ms, the number of pulses is 10, the electroporation transfection is completed, the human embryonic stem cells are put back into the cell culture solution for culture, and the fluorescence in the cells is observed to judge the introduction/transfection efficiency.
Fig. 5a is a photograph of green fluorescence of primary human PBMC cells under 488nm laser after introducing FAM-labeled siRNA fragments into the cells for 5 minutes, and fig. 5b is a photograph of the cells in fig. 5a under bright field, and it can be seen from fig. 5a-5b that the cells are in good condition after electroporation and the introduction efficiency is high (greater than 70%).
Example 3
The present invention provides a method for introducing an Alexa Fluor 488-labeled H4B4 monoclonal antibody into HeLa cells, specifically comprising:
using the apparatus shown in fig. 2, HeLa cells were cultured to logarithmic growth phase, and a H4B4 monoclonal antibody labeled with an exogenous substance Alexa Fluor 488 was formulated into a solution having a concentration of 80 μ g/mL with sterile water;
the cells in the logarithmic phase of growth were placed in the apparatus shown in FIG. 1, 100. mu.L of the solution containing Alexa Fluor 488-labeled H4B4 monoclonal antibody was placed in the second storage unit, and the substrate on which the HeLa cells were placed was immersed in the solution containing Alexa Fluor 488-labeled H4B4 monoclonal antibody, and the HeLa cells were not contacted with the solution containing Alexa Fluor 488-labeled H4B4 monoclonal antibody.
Controlling the touch display unit to generate a first pulse signal via the pulse switch unit, wherein the first voltage U is higher than the first voltage1Is 3V, the first pulse width is 10ms, and a first current I is obtained1 198.5mA；
According to a first current I1Sample size and cell density the first state was determined to be StL2 and the resistance threshold was determined to be 35 Ω A according to tables 2-3-1Step voltage Ustep0.75V;
the touch display unit controls the pulse switch unit to generate a second pulse signal, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2At 4V, with a step voltage UstepGradually increasing, the pulse width is 5ms, and the pulse interval is 5 ms;
when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3And when the resistance change rate of the first system is equal to the resistance threshold value, a third voltage U is applied3The touch display unit controls the pulse switch unit to generate a second pulse signal, a third voltage U37.75V, a third pulse width of 10ms, a pulse interval of 10ms and 10 pulses, and the electroporation transfection is completed, the HeLa cells are put back into the cell culture solution for culture, and the fluorescence in the cells is observed to judge the introduction/transfection efficiency.
Fig. 6a is a photograph of green fluorescence of HeLa cells introduced with Alexa Fluor 488-labeled H4B4 monoclonal antibody for 5 minutes after the cells were irradiated with 488nm laser, and fig. 6B is a photograph of the cells provided in fig. 6a under bright field, and it can be seen from fig. 6a-6B that the cells were in good condition after electroporation and the introduction efficiency was high (more than 70%).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An apparatus for introducing exogenous material into a cell, comprising a reaction module and a control module;
the reaction module comprises a first storage unit, a second storage unit and electrodes, wherein the first storage unit comprises a substrate provided with a plurality of through holes, the substrate is used for placing cells, the second storage unit is communicated with the through holes and used for placing the exogenous substances, and the electrodes are respectively connected with the first storage unit and the second storage unit;
the control module is connected with the electrode and used for controlling the electrode to generate a pulse signal.
2. The device of claim 1, wherein the through-holes have a pore size of 50nm to 8 μm and a height of 5 μm to 50 μm, and the density of the through-holes on the substrate is 1 x 104Per cm2-4*108Per cm2。
3. The apparatus of claim 1, wherein the substrate comprises a silicon-containing inorganic substance selected from one or more of elemental silicon, silicon oxide, and silicon nitride, or a polymer selected from one or more of polycarbonate, polyethylene terephthalate, and polyimide.
4. The device of claim 1, wherein the electrodes comprise a positive electrode and a negative electrode, the positive electrode being connected to the first storage unit and the negative electrode being connected to the second storage unit.
5. The device according to any one of claims 1 to 4, wherein the control module comprises a microcontroller unit, and a voltage generation unit, a current detection unit and a pulse switch unit connected with the microcontroller unit;
the voltage generating unit is used for receiving signals of the microcontroller unit and sending voltage, the voltage generating unit is connected with the current detecting unit, the current detecting unit is connected with the pulse switch unit, the current detecting unit is used for detecting current of the reaction module and feeding back the current to the microcontroller unit, and the pulse switch unit is connected with the electrode and used for receiving signals of the microcontroller unit and controlling the on-off of the pulse signals.
6. The apparatus of claim 1, comprising a power module to provide power to the control module.
7. The device of claim 1, comprising a touch display module, wherein the touch display module is connected to the control module for human-computer interaction.
8. The device of claim 1, comprising a sample holder comprising a wall formed by the enclosure, the wall being provided with a first projection for carrying the first storage unit and a second projection for carrying the second storage unit.
9. A method for introducing exogenous material into a cell, using the device of any one of claims 1-8, comprising the steps of:
placing the cells in the logarithmic growth phase on the surface of the substrate, placing the solution containing the exogenous substances into the second storage unit, and immersing part of the substrate into the solution containing the exogenous substances, wherein the cells are not in contact with the solution containing the exogenous substances, so as to obtain a reaction system;
and electrifying the reaction system to enable the exogenous substances to enter the cells through the through holes under a pulse signal.
10. The method of claim 9, wherein the pulse signal comprises a first pulse signal, a second pulse signal, and a third pulse signal, wherein:
generating a first pulse signal by a control module, wherein a first voltage U of the first pulse signal11-3V, the width of the first pulse signal is 5-20ms, and the cell is obtained at a first voltage U1First current I of1And a first resistor R1；
According to the first current I1And determining a first state from the number of cells, and determining a threshold value RI and a step voltage U from the first statestep；
Generating a second pulse signal by the control module, wherein the second pulse signal is at a second voltage U2Is an initial voltage, the second voltage U2Greater than a first voltage U1By step voltage UstepGradually increasing, the width of the second pulse signal is 5-20ms, and the interval of the second pulse signal is 5-20 ms;
when the second voltage U is applied2By step voltage UstepGradually increases to a third voltage U3Generating a third pulse signal when the change rate delta RI of the resistance of the cell along with the current is in a decreasing trend and is less than or equal to a threshold value RI, wherein the voltage of the third pulse signal is a third voltage U3The width of the third pulse signal is 10-50ms, the interval of the third pulse signal is 10-50ms, and the number of the third pulse signals is 1-20And completing the electrifying process.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826408A (en) * | 2003-03-14 | 2006-08-30 | 塞托·帕尔斯科技公司 | Large volume ex vivo electroporation method |
| CN110452807A (en) * | 2019-07-10 | 2019-11-15 | 北京航空航天大学 | A kind of biochip and its preparation method and application to cell delivering target molecule |
| CN112226365A (en) * | 2020-10-13 | 2021-01-15 | 北京航空航天大学 | Nano-electroporation device based on single cell array and application thereof |
-
2021
- 2021-09-08 CN CN202111048623.2A patent/CN113832192A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826408A (en) * | 2003-03-14 | 2006-08-30 | 塞托·帕尔斯科技公司 | Large volume ex vivo electroporation method |
| CN110452807A (en) * | 2019-07-10 | 2019-11-15 | 北京航空航天大学 | A kind of biochip and its preparation method and application to cell delivering target molecule |
| CN112226365A (en) * | 2020-10-13 | 2021-01-15 | 北京航空航天大学 | Nano-electroporation device based on single cell array and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LINGQIAN CHANG: "3D Nanochannel Array for High-Throughput Cell Manipulation and Electroporation", 《METHODS MOL BIOL .》, vol. 2050, pages 29 - 41 * |
| LINGQIAN CHANG: "3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control", 《NANOSCALE》, vol. 8, no. 1, pages 243 - 252, XP055790463, DOI: 10.1039/C5NR03187G * |
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