CN118147127B - Large-scale continuous electrotransfection method and system - Google Patents
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Abstract
The application relates to a large-scale continuous electrotransfection method and a system, wherein the method adopts the following steps: applying a constant voltage to the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells are subjected to voltage change due to movement when passing through the three-section electric field area and are subjected to pulse electric shock; by applying the method provided by the application, the constant voltage is applied to the electrode of the electrotransfer tube, so that the cell suspension can complete the electrotransfer process in the whole continuous motion process of the electric field, thereby being more suitable for the electrotransfer mode of a closed pipeline in GMP (good manufacturing practice) standard, realizing high-flux and large-scale electrotransfer, improving the transfer efficiency and improving the transfer quality.
Description
Technical Field
The invention relates to the technical field of electrotransfection, in particular to a large-scale continuous electrotransfection method and a system.
Background
Electrotransfection systems are devices that use an electric field to introduce foreign substances (e.g., DNA, RNA, etc.) into cells, and the art of which mainly relates to many disciplines such as cell biology, molecular biology, biotechnology, etc. Electrotransfection systems are widely used in basic research, biopharmaceuticals, agricultural technology, immunotherapy, stem cell research, neuroscience research, metabolic disease research, and the like. Electrotransfection systems serve an essential biotechnology tool, playing an indispensable role in a variety of fields;
Cell therapy plays an important role in the development of medical health as innovative biotechnology, and as more and more products go to the market, the number of clinical trials grows exponentially, and the industry has come to a key period of high-speed growth. Many cell therapy products have been successfully commercialized.
Among the various innovative therapies, such as CAR-T, CAR-NK, TIL, TCR-T, how to efficiently perform cell engineering is one of the main core technologies. Gene transduction using viruses is currently the mainstream modification method, however, non-viral technology has advantages of better safety, simpler process flow, etc., gradually attracts more and more developers, and has potential to become the main trend of next generation cell and gene therapy.
Compared with the virus technology, the non-virus cell transformation technology has the advantages of short preparation time, easy storage and low cost in terms of production and manufacture; in terms of reconstruction strategies, DNA, RNA and protein can be flexibly introduced into the non-virus vector according to requirements; and can realize the fixed-point insertion of genes, ensure the accuracy and the controllability to minimize the safety risk. In addition, the non-viral gene transduction technology provides a new technology support for novel therapies such as general CAR-T, CAR-NK, iPSC-derived cell therapies and the like.
The non-viral gene transduction technology in cell and gene therapy has two main routes, one is transposon, and the other is site-directed integrated gene editing based on CRISPR/Cas 9. Non-viral gene transduction techniques require the introduction of exogenous genes into a large number of cells by an efficient method.
Electroporation has proven to be very promising for non-viral transfection and has been one of the most widely used techniques; most of the electrotransfection is only suitable for small-scale research at present, and clinical application needs to be compatible with treatment scale and speed, stable efficiency and higher cell activity, and compliance of the whole production process. Therefore, the result of the small-scale electrotransfection experiment is amplified to the clinical production scale, and the bottleneck which restricts the development result to push to the practical industrial application is formed.
The existing domestic and foreign cell electroporation transfection technical routes adopt a pulse electric field mode to carry out electroporation transfection on a fixed-volume cell sample, a high-flux cell electroporation transfection process can be realized only through a separate transfection mode, CN 110903970A-an intermittent flow type electrotransfection device, CN 110885857A-an intermittent flow type electrotransfection device, CN 109679844A-a flow type electrotransfection device and the like, and particularly CN 109679844A-a flow type electrotransfection device, although the scheme is stated to be capable of carrying out continuous electrotransfection, a high-voltage pulse power supply is adopted, the consistent electric shock perforation operation on continuously sampled cells cannot be realized, the effect obtained in practice is still unsatisfactory, and a large-scale continuous electrotransfection method and system capable of overcoming the defects are needed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a large-scale continuous electrotransfection method and a large-scale continuous electrotransfection system aiming at the defects of the prior art.
The technical scheme adopted for solving the technical problems is as follows:
A large-scale continuous electrotransfection method is constructed, and the method comprises the following steps:
applying a constant voltage to the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells undergo a voltage change due to motion while passing through the three-stage electric field region, and are subjected to a pulsed electric shock.
The invention relates to a large-scale continuous electrotransfection method, wherein the generation of an electric field adopts the following steps:
two flat plate electrodes are arranged at the non-end part of the electrotransport tube;
a constant voltage is applied to both of the plate electrodes to shape the inflow region, the working region, and the outflow region within the electrotransport tube.
The invention relates to a large-scale continuous electrotransfection method, wherein the electrotransfection tube is arranged by the following steps:
A square tubular pipe body is selected, two ends of the pipe body are respectively provided with a liquid inlet and a liquid outlet, and the inner wall of the pipe body is insulated; the two opposite side surfaces of the middle part of the tube body are provided with the flat plate electrodes.
The large-scale continuous electrotransfection method of the present invention, wherein the method further comprises the steps of:
And applying a constraint magnetic field to the inflow region and the outflow region of the electrotransfer tube, and controlling the electric field range and the electric field intensity of the inflow region and the outflow region through the constraint magnetic field to control the pulse waveforms of the ascending section and the descending section of the electric shock pulse born by the cells.
The large-scale continuous electrotransfection method of the present invention, wherein the method further comprises:
The width of the electric shock pulse born by the cells is adjusted by controlling the flow rate of the cell suspension flowing through the electrotransport tube.
The large-scale continuous electrotransfection method of the present invention, wherein the method further comprises:
the cell suspension in the electrotransfer tube adopts continuous sample injection.
The large-scale continuous electrotransfection method of the invention, wherein the confining magnetic field is perpendicular to the direction of the electric field.
A large-scale continuous electrotransfection system employing a large-scale continuous electrotransfection method as described above, wherein the system comprises an electrotransfection tube and an electric field unit;
the electric field unit is used for applying constant voltage on the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells undergo a voltage change due to motion while passing through the three-stage electric field region, and are subjected to a pulsed electric shock.
The invention relates to a large-scale continuous electrotransfection system, wherein the system further comprises a magnetic field unit; the magnetic field unit is used for applying a constraint magnetic field to the inflow region and the outflow region of the electrotransfer tube, controlling the electric field range and the electric field intensity of the inflow region and the outflow region through the constraint magnetic field, and controlling pulse waveforms of the ascending section and the descending section of electric shock pulses born by cells.
The invention relates to a large-scale continuous electrotransfection system, wherein the system further comprises a flow rate control unit;
The flow rate control unit is used for controlling the flow rate of the cell suspension flowing through the electrotransfer tube and adjusting the breadth of electric shock pulse born by the cells; the cell suspension in the electrotransfer tube adopts continuous sample injection.
The application has the beneficial effects that: by applying the method provided by the application, the constant voltage is applied to the electrode of the electrotransfer tube, so that the cell suspension can complete the electrotransfer process in the whole continuous motion process of the electric field, thereby being more suitable for the electrotransfer mode of a closed pipeline in GMP (good manufacturing practice) standard, realizing high-flux and large-scale electrotransfer, improving the transfer efficiency and improving the transfer quality.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described with reference to the accompanying drawings and embodiments, in which the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained by those skilled in the art without inventive effort:
FIG. 1 is a flow chart of a large-scale continuous electrotransfection method according to a preferred embodiment of the present invention;
FIG. 2 is a schematic view of the equipotential surface of an open type constant voltage electrotransfection chamber of the large-scale continuous electrotransfection method according to the preferred embodiment of the present invention;
FIG. 3 is a schematic illustration of the equipotential surfaces of a large-scale continuous electrotransfection method according to a preferred embodiment of the present invention;
FIG. 4 is a schematic diagram of the flow rate electric pulse structure of the electrotransport tube of the large-scale continuous electrotransfection method according to the preferred embodiment of the present invention;
FIG. 5 is a schematic diagram of the electric field of a plate electrode of a large-scale continuous electrotransfection method according to a preferred embodiment of the present invention;
FIG. 6 is a flow chart of a large-scale continuous electrotransfection method according to another preferred embodiment of the present invention;
FIG. 7 is a schematic diagram of the electric field of the electrotransfer chamber of the large-scale continuous electrotransfection method according to another preferred embodiment of the present invention;
FIG. 8 is a schematic block diagram of a large-scale continuous electrotransfection system in accordance with a preferred embodiment of the present invention.
Reference numeral 1 is an electrode; 2 is an equipotential surface; 3 is a sample inflow port; 4 is an insulating pipe wall; 5 is an electric shock cavity; 6 is a sample outflow port; 7 is the magnetic field of the inflow region; 8 is the magnetic field of the outflow region; 9 is the electric field line after constraint; u is voltage; uw is the operating voltage; l is an electric shock zone; lin is the inflow region; lw is the working area; lout is the outflow zone; v is the sample flow rate; t is the electric shock time; lo is the electric field range.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following description will be made in detail with reference to the technical solutions in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
1. Embodiment one:
The large-scale continuous electrotransfection method according to the preferred embodiment of the present invention, as shown in FIG. 1, adopts:
s01: applying a constant voltage to the electrode of the electrotransport tube;
S02: dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened;
s03: the cells are subjected to voltage change due to movement when passing through the three-section electric field area and are subjected to pulse electric shock;
By applying the method provided by the application, the constant voltage is applied to the electrode of the electrotransfer tube, so that the cell suspension can complete the electrotransfer process in the whole continuous motion process of the electric field, thereby being more suitable for the electrotransfer mode of a closed pipeline in GMP (good manufacturing practice) standard, realizing high-flux and large-scale electrotransfer, improving the transfer efficiency and improving the transfer quality.
As shown in fig. 2, the invention designs a hollow square tube-shaped electrotransport tube with two open ends, which is composed of two parts consisting of a pair of flat plate electrodes 1 and an insulating tube wall 4; the flat plate electrodes 1 are arranged on the pipe walls at two opposite sides in parallel, the inner sides of the electrodes are exposed in the pipe to generate an electric shock field, and the outer sides of the electrodes are exposed outside the pipe to be connected with an external high-voltage generator. Two ends of the square tube are provided with liquid tube connectors for the inflow and outflow of the electrokinetic transfer sample liquid.
A constant voltage is applied to the plate electrode 1, and an electric field is formed in the square tube-shaped electrotransport tube as shown in fig. 2 and 3. The flat electrode area is an electric shock cavity 5, and has a uniform electric field; the sample inlet 3 and sample outlet 6 portions of the open areas at both ends have a gradient electric field from weak to strong due to the distance from the electrode.
The electric field in the electrotransport tube is in a steady state with a steady voltage across the planar electrode. The electrotransfection process of the present invention, electrotransport tubes, always operate under such steady state conditions.
The invention realizes the pulse electric shock perforation of sample cells by controlling the sample flow rate of the cell suspension flowing through the electrotransport tube on the basis of the electrotransport tube, thereby achieving the purpose of cell transfection.
As shown in fig. 4, a set working voltage U is applied to the electrode of the electrotransport tube, and stable three-section electric field regions of increasing, decreasing and decreasing are formed in the inlet region Lin, the working region Lw and the outlet region Lout of the electrotransport tube.
The cell dynamically passes through the three electric field regions, and is subjected to a voltage 7 pulse as shown in the upper part of fig. 4, wherein the voltage rising section of the voltage pulse corresponds to the cell sample inlet region Lin of the electrotransport tube, the voltage holding section of the electric pulse corresponds to the cell sample working region Lw, and the voltage falling section of the electric pulse corresponds to the cell sample outlet region Lout. The amplitude of the electric shock pulse can be adjusted by adjusting the working voltage U, and the amplitude of the electric shock pulse can be adjusted by adjusting the flow velocity of the sample suspension, so that the adjustment of the cell electric transfection electric shock parameters is realized.
The working voltage is kept stable without changing the electric field state in the whole transfection process; the whole electroporation transfection process is a continuous sample injection process of a cell sample, the transfection flux is positively related to the through-flow section, the through-flow speed and the through-flow time of the electrotransport tube, and the high-flux continuous electroporation transfection of cells can be easily realized by the scheme of the invention; the electric shock duration T of the cell sample is inversely proportional to the sample flow rate due to the fixation of the electric shock region, and the duration of the cell pulse electric shock can be precisely controlled by controlling the sample flow rate.
Unlike fixed sample volume, electrotransfection methods of electrical pulses, the cell sample transfection process of the present invention is a continuous high-throughput cell electroporation transfection system of controlled constant voltage.
Adopting a continuously flowing sample conductive liquid to reduce electrolytic reaction of unit liquid, thereby reducing bubble generation;
As shown in fig. 5, the intensity of the electric field of the electrotransport tube can be controlled by adjusting the magnitude of the voltage applied to the electrode; the shape of the electric field of the electrotransport tube can be appropriately adjusted by adjusting the shape of the electrode.
2. Embodiment two:
The method for large-scale continuous electrotransfection according to another preferred embodiment of the present invention is shown in FIG. 6, and referring to FIG. 7, and is similar to the first embodiment, except that the following description is omitted: the method comprises the following steps:
s11: applying a constant voltage to the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells are subjected to voltage change due to movement when passing through the three-section electric field area and are subjected to pulse electric shock;
S12: applying a constraint magnetic field to an inflow region and an outflow region of the electrotransfer tube, controlling the electric field range and the electric field intensity of the inflow region and the outflow region through the constraint magnetic field, and controlling pulse waveforms of an ascending section and a descending section of electric shock pulses born by cells;
By applying the method of the application, a constraint magnetic field is added to the inflow region and the outflow region of the electric field coverage region, and the electric field of the inflow region and the outflow region is converged and diverged by the constraint magnetic field, so that the range and the intensity of the electric field are changed, and the control of pulse waveforms of the ascending section and the descending section of the electric shock pulse born by the cells is realized; by adopting the method, the whole section of the electric field can be controlled as required, and the pulse waveform is designed by the method, so that the cell suspension is in the electric transfection area in the whole process of passing through the electric field, thereby achieving the aim of improving the transfection efficiency.
As shown in fig. 7, the invention sets a restraining magnetic field in the inflow area and the outflow area of the electrotransport tube, and the electric field shape and strength of the inflow area and the outflow area of the electrotransport tube can be dynamically adjusted by applying the restraining magnetic field perpendicular to the electric field.
It is known from electromagnetic principles that a magnetic field produces a force on the charge distribution in an electrode.
When a magnetic field is present in the electrode field, the magnetic field will exert a force on the charge distribution in the electrode, causing the charge to redistribute inside the electrode, changing the distribution of the electrode electric field.
The process of electrode-to-cell electrical shock is established by the directional movement of the charge. The lorentz force exerted by the charged particles in the magnetic field acts both perpendicularly to the direction of the magnetic field and perpendicularly to the direction of movement of the charge. The lorentz force is always perpendicular to the moving speed direction of the charged particles, and cannot change the moving speed of the charged particles, but only changes the moving speed direction.
The magnetic field perpendicular to the electric field may control the convergence and divergence of the regional electric field, thereby changing the extent and strength of the electric field.
According to the principle, the invention controls the electric field range and the electric field intensity of the inflow region and the outflow region through the magnetic field, thereby realizing the control of pulse waveforms of the ascending section and the descending section of the electric shock pulse born by the cells.
3. Embodiment III:
a large-scale continuous electrotransfection system, using a large-scale continuous electrotransfection method as described above, as shown in fig. 8, comprises an electrotransfection tube 100 and an electric field unit 102;
An electric field unit 102 for applying a constant voltage to an electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells are subjected to voltage change due to movement when passing through the three-section electric field area and are subjected to pulse electric shock;
The constant voltage is applied to the electrode of the electrotransfection tube, so that the cell suspension can complete the electrotransfection process in the whole continuous motion process of an electric field, thereby being more suitable for the electrotransfection mode of a closed pipeline in GMP (good manufacturing practice) standard, realizing high-flux and large-scale electrotransfection, improving the transfection efficiency and improving the transfection quality;
the system may further comprise a magnetic field unit 101; a magnetic field unit 101 for applying a confining magnetic field to the inflow region and the outflow region of the electrotransport tube, controlling the electric field range and intensity of the inflow region and the outflow region by the confining magnetic field, and controlling pulse waveforms of the ascending and descending sections of the electric shock pulse received by the cells;
the restriction magnetic field is added to the inflow region and the outflow region of the electric field coverage region, and the electric field of the inflow region and the outflow region is converged and diverged by the restriction magnetic field, so that the range and the intensity of the electric field are changed, and the control of pulse waveforms of the ascending section and the descending section of the electric shock pulse born by the cells is realized; by adopting the method, the whole section of the electric field can be controlled as required, and the pulse waveform is designed by the method, so that the cell suspension is in the electric transfection area in the whole process of passing through the electric field, thereby achieving the aim of improving the transfection efficiency.
Preferably, the system further comprises a flow rate control unit 103; and a flow rate control unit 103 for controlling the flow rate of the cell suspension flowing through the electrotransport tube and adjusting the breadth of the electric shock pulse.
It will be understood that modifications and variations will be apparent to those skilled in the art from the foregoing description, and it is intended that all such modifications and variations be included within the scope of the following claims.
Claims (8)
1. A large-scale continuous electrotransfection method, which is characterized by comprising the following steps:
Applying a constant voltage to the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells are subjected to voltage change due to movement when passing through the three-section electric field area and are subjected to pulse electric shock;
Applying a constraint magnetic field to an inflow region and an outflow region of the electrotransfer tube, controlling the electric field range and the electric field intensity of the inflow region and the outflow region through the constraint magnetic field, and controlling pulse waveforms of an ascending section and a descending section of electric shock pulses born by cells;
The generation method of the electric field comprises the following steps:
two flat plate electrodes are arranged at the non-end part of the electrotransport tube;
a constant voltage is applied to both of the plate electrodes to shape the inflow region, the working region, and the outflow region within the electrotransport tube.
2. The method of large-scale continuous electrotransfection according to claim 1, wherein the electrotransfer tube setup employs the method of:
A square tubular pipe body is selected, two ends of the pipe body are respectively provided with a liquid inlet and a liquid outlet, and the inner wall of the pipe body is insulated; the two opposite side surfaces of the middle part of the tube body are provided with the flat plate electrodes.
3. The large-scale continuous electrotransfection method of claim 1, further comprising:
The width of the electric shock pulse born by the cells is adjusted by controlling the flow rate of the cell suspension flowing through the electrotransport tube.
4. The large-scale continuous electrotransfection method of claim 1, further comprising:
the cell suspension in the electrotransfer tube adopts continuous sample injection.
5. The method of claim 1, wherein the confining magnetic field is perpendicular to the direction of the electric field.
6. A large scale continuous electrotransfection system employing a large scale continuous electrotransfection method according to any one of claims 1 to 5, characterized in that the system comprises an electrotransfection tube and an electric field unit;
the electric field unit is used for applying constant voltage on the electrode of the electrotransport tube; dividing an electric shock section covered by an electric field in an electric rotating tube into an inflow area, a working area and an outflow area along the flowing direction of cell suspension; the inflow region, the working region and the outflow region are respectively three sections of electric field regions which are gradually increased, evenly increased and weakened; the cells undergo a voltage change due to motion while passing through the three-stage electric field region, and are subjected to a pulsed electric shock.
7. The large-scale continuous electrotransfection system of claim 6, wherein the system further comprises a magnetic field unit; the magnetic field unit is used for applying a constraint magnetic field to the inflow region and the outflow region of the electrotransfer tube, controlling the electric field range and the electric field intensity of the inflow region and the outflow region through the constraint magnetic field, and controlling pulse waveforms of the ascending section and the descending section of electric shock pulses born by cells.
8. The large scale continuous electrotransfection system of claim 7, wherein the system further comprises a flow rate control unit;
The flow rate control unit is used for controlling the flow rate of the cell suspension flowing through the electrotransfer tube and adjusting the breadth of electric shock pulse born by the cells; the cell suspension in the electrotransfer tube adopts continuous sample injection.
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| US20090081750A1 (en) * | 2007-02-14 | 2009-03-26 | Bio-Rad Laboratories, Inc. | Transfection in magnetically driven continuous flow |
| WO2016003485A1 (en) * | 2014-07-03 | 2016-01-07 | Massachusetts Institute Of Technology | Microfluidic assay for rapid optimization of cell electroporation |
| CN116987589A (en) * | 2017-10-19 | 2023-11-03 | 苏州壹达生物科技有限公司 | A flow electroporation device |
| CA3138353A1 (en) * | 2019-05-16 | 2020-11-19 | CyteQuest, Inc. | Devices, methods, and systems for electroporation |
| JP7691981B2 (en) * | 2019-11-22 | 2025-06-12 | ザ チャールズ スターク ドレイパー ラボラトリー， インク． | System for electroporation of particles |
| CN114686371A (en) * | 2020-12-29 | 2022-07-01 | 苏州壹达生物科技有限公司 | A flow electrotransfection device |
| CN112980673B (en) * | 2021-02-01 | 2023-03-24 | 重庆大学 | High-frequency pulse magnetic field induced cell magnetic perforation device and method |
| CN112980674A (en) * | 2021-02-01 | 2021-06-18 | 重庆大学 | Device and method for enhancing high-frequency pulse magnetic field to induce magnetic perforation of cells based on targeted modified magnetic nanoparticles |
| JP2024515808A (en) * | 2021-04-27 | 2024-04-10 | カイトペン コーポレイション | Methods of electromechanical transfection |
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