WO2024012261A1 - 一种流式电穿孔装置 - Google Patents

一种流式电穿孔装置 Download PDF

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Publication number
WO2024012261A1
WO2024012261A1 PCT/CN2023/104615 CN2023104615W WO2024012261A1 WO 2024012261 A1 WO2024012261 A1 WO 2024012261A1 CN 2023104615 W CN2023104615 W CN 2023104615W WO 2024012261 A1 WO2024012261 A1 WO 2024012261A1
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WIPO (PCT)
Prior art keywords
side wall
electroporation device
flow electroporation
electrode assembly
working state
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Application number
PCT/CN2023/104615
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English (en)
French (fr)
Inventor
戴晓兵
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苏州壹达生物科技有限公司
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Publication of WO2024012261A1 publication Critical patent/WO2024012261A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a flow electroporation device.
  • Electroporation is a technology that uses an electric field to act on the cell membrane to create micropores in the cell membrane that can allow foreign molecules such as DNA, RNA, and proteins to pass through. These micropores are generated instantly under the action of the electric field and can be formed at any time. The electric field disappears and is restored, thereby avoiding permanent damage to cells. Electroporation technology can be used to introduce DNA, RNA, proteins, sugars, dyes, virus particles and other foreign molecules into prokaryotic or eukaryotic cells, which is called electrotransfection. Electroporation technology is used in antibody proteins. It is widely used in many high-end biomedical technology fields such as production, in vitro diagnostic (IVD) reagent raw material production, immune cell gene editing and gene modification, and is an important technology for intracellular material delivery.
  • IVD in vitro diagnostic
  • a common flow electroporation device mainly includes a pair of planar electrodes used to generate an electric field, and a chamber located between the two planar electrodes.
  • the cell suspension can receive an electric shock while flowing through the chamber, resulting in electroporation, and thus the cells Foreign molecules in suspension can enter target cells.
  • most flow electroporation devices have problems such as low cell processing efficiency and large residual amounts of cell suspension.
  • the purpose of the present invention is to provide a flow electroporation device with high cell processing efficiency in view of the problems existing in the prior art.
  • a flow electroporation device includes a host, an electrode assembly, a sampling container and a collection container, the electrode assembly, the sampling container and the collection container are respectively provided on the host ,in,
  • the electrode assembly includes an electrode used for electroporation of cell suspension, the electrode assembly has a liquid inlet and a liquid outlet, and the electrode assembly can be relatively rotatably arranged on one side of the host;
  • the sampling container is used to store the cell suspension to be processed by the electrode, and the sampling container is connected to the liquid inlet through a liquid inlet pipeline;
  • the collection container is used to receive the cell suspension treated by the electrode, and the collection container is connected to the liquid outlet through a liquid outlet pipeline;
  • the flow electroporation device has a first working state and a second working state.
  • the electrode When the flow electroporation device is in the first working state, the electrode performs electroporation on the cell suspension; when the flow electroporation device is in the first working state, During the process of the flow electroporation device converting from the first working state to the second working state, the electrode assembly rotates relative to the host; when the flow electroporation device is in the second working state , at least part of the cell suspension in the electrode assembly can flow out from the liquid outlet under the action of gravity.
  • the height of the liquid inlet is lower than the height of the liquid outlet; when the flow electroporation device is in the In the second working state, the height of the liquid outlet is reduced.
  • the flow electroporation device can operate normally.
  • the cell suspension continues to flow through the electrode assembly in an inward and upward direction and is electroporated. Under the action of the gravity of the cell suspension itself, it can fill the electrode pads.
  • the chambers between the electrodes maintain electrical communication between the electrode sheets, improve the continuity and uniformity of the liquid flow, effectively reduce the generation of bubbles, and improve the uniformity of the electric field, thus comprehensively improving the electroporation efficiency.
  • the height of the liquid outlet is reduced so that the unexhausted cell suspension in the chamber can be discharged under its own gravity.
  • the height of the liquid outlet is lower than the height of the liquid inlet.
  • the electrode assembly has a larger tilt angle, which helps the cell suspension to be discharged more fully.
  • the axis line of the liquid inlet and the axis line of the liquid outlet both extend in the vertical direction. Therefore, when in the first working state, the cell suspension flows vertically upward through the electrode assembly, which can better ensure the uniformity of the liquid flow and the uniformity of the electric field.
  • the electrodes include a first electrode and a second electrode arranged at intervals, with a processing channel between the first electrode and the second electrode, and the flow electroporation device is in the first In the working state, the processing channel extends in the up and down direction, and the cell suspension flows through the processing channel from bottom to top. Therefore, the cell suspension not only flows in and out of the inlet and outlet of the entire electrode assembly, but also maintains the flow direction of bottom in and out in the specific processing channel between the electrodes, further ensuring that the flow direction of the cell suspension in the chamber between the electrodes is Liquid flow uniformity and electric field uniformity.
  • the flow electroporation device further includes a liquid level sensor disposed between the sampling container and the electrode assembly, and the liquid level sensor is fixed on one side of the host machine.
  • the height of the liquid level sensor is lower than the height of the electrode assembly.
  • the liquid level sensor has a pipeline guide groove that can closely fit the liquid inlet pipeline, which is beneficial to the normal operation of the liquid level sensor.
  • the pipeline guide groove extends along the vertical direction, and the liquid inlet pipeline passes through the pipeline guide groove from bottom to top.
  • the liquid level sensor can be used to measure the liquid level status in the liquid inlet pipeline and transmit the signal to the control system, which controls the working status of the electrode assembly.
  • the liquid level sensor is placed vertically and the cell suspension in the sampling line moves in and out.
  • This method can ensure that the liquid inlet pipeline in the pipeline guide tank is filled with liquid, preventing the liquid in the pipeline from being dissatisfied or air bubbles causing misjudgment of the test results and affecting the correct control of the electroporation process by the control system.
  • the flow electroporation device further includes a peristaltic pump, which is fixedly installed on one side of the host machine, and the liquid outlet pipeline passes through the peristaltic pump from top to bottom.
  • the peristaltic pump is used to provide the power for the cell suspension to flow throughout the flow electroporation device.
  • the peristaltic pump is set between the collection container and the electrode assembly, and is installed at the rear end and below of the electrode assembly. The peristaltic pump and the electrode assembly The liquid outlet pipeline is more smoothly integrated.
  • the peristaltic pump When the peristaltic pump is started, the negative pressure formed in the electrode assembly and the pipeline on its front side draws the cell suspension out of the sampling container; when the entire pipeline is filled with liquid, the internal pressure of the pipeline reaches equilibrium and peristalsis The pump transports the cell suspension stably.
  • the flow rate of the cell suspension in the present invention is more uniform and stable, which helps to obtain more accurate results. Good electroporation treatment effect.
  • the flow electroporation device further includes a liquid level sensor and a peristaltic pump.
  • the host computer has a first side wall, and a second side located on two sides with different width directions of the first side wall. wall and a third side wall, the extension direction of the first side wall intersects the extension direction of the second side wall, and the extension direction of the first side wall intersects the extension direction of the third side wall;
  • the electrode assembly is relatively rotatably disposed on the first side wall, the liquid level sensor and the peristaltic pump are disposed on the first side wall; the sampling container is disposed on the second On the side wall, the collection container is provided on the third side wall.
  • the first side wall is perpendicular to the second side wall, and the first side wall and the third side wall are also perpendicular.
  • the liquid level sensor, electrode assembly, and peristaltic pump are placed on the same side wall.
  • the pipelines are arranged more neatly.
  • the rotation of the electrode assembly will not easily cause cross-winding of the pipelines, ensuring that the liquid level sensor, electrode assembly, and peristaltic pump are arranged on the same side wall.
  • the flow is smooth; the sampling container and the collection container are respectively located on the left and right side walls (i.e. the second side wall and the third side wall). Their positions are relatively fixed and are not easily affected by the rotation of the electrode assembly. It also helps to shorten the width of the host and reduce the cost. Small host takes up space.
  • the flow electroporation device further includes a rotating base and a rotating driving device.
  • the rotating base can be relatively rotatably connected to the host machine around the rotation center line.
  • the rotating driving device is used to drive the The rotating base rotates, and the electrode assembly is detachably arranged on the rotating base and can rotate synchronously with the rotating base.
  • the rotating base helps to improve the stability and reliability of the electrode assembly when rotating.
  • the electrode assembly and the rotating base move synchronously.
  • the rotation center line extends in a horizontal direction, and there is a baseline.
  • the baseline is a ray perpendicular to the rotation center line and extending vertically upward.
  • the endpoint of the baseline is located on the rotation center line.
  • the direction in which the electrode assembly rotates from the reference position to the second limit position is Positive direction; when the electrode assembly is in the reference position, the axis line of the liquid outlet coincides with the baseline; when the electrode assembly is in the first extreme position, the liquid outlet The angle between the axis of the axis and the baseline is -10°; when the electrode assembly is in the second At the extreme position, the angle between the axis of the liquid outlet and the baseline is 190°.
  • a first limiting structure is provided between the rotating base and the main machine, and the first limiting structure is used to limit the electrode assembly between the first limit position and the second Rotate between extreme positions.
  • the first limiting structure limits the rotation angle of the electrode assembly, and the electrode assembly can only rotate between preset limit positions.
  • the electrode assembly in the first working state, the electrode assembly is in the reference position; in the second working state, the angle between the axis of the liquid outlet and the baseline is approximately 150°.
  • the pipeline near the liquid outlet of the electrode assembly The remaining cell suspension can be drained better.
  • the first limiting structure effectively avoids the pulling or knotting of the pipeline that may be caused by an excessive rotation angle, thereby improving the reliability of the operation of the flow electroporation device.
  • the main machine is provided with a support plate extending in the up and down direction, and a limited hole is provided on the support plate;
  • the rotation base has a connection part, and the rotation center line passes through the connection part, so
  • the connecting part has a convex part protruding outward along the radial direction of the rotation center line, and the connecting part is inserted into the limiting hole;
  • the first limiting structure includes the convex part and the Limiting hole, along the circumferential direction of the rotation center line, the side wall of the limiting hole has a first boss and a second boss arranged at intervals; when the electrode assembly is in the first limit position, The convex part abuts the first boss; when the electrode assembly is in the second extreme position, the convex part abuts the second boss.
  • the first limiting structure specifically adopts a structure in which a simple convex part matches two sets of bosses, which has a simple design, does not occupy space, and has strong reliability.
  • the main machine has a first side wall, and the rotating base is relatively rotatably connected to the first side wall around the rotation center line, and the rotation center line is perpendicular to the first side wall.
  • the rotating seat includes a rotating platform. Along the extension direction of the rotating center line, a gap is provided between the rotating platform and the first side wall.
  • the liquid inlet and the liquid outlet are both Located on a side of the rotating platform away from the first side wall.
  • the rotating table provides a support plane for the rotation of the electrode assembly.
  • the rotating table maintains a movement gap of about 0.5 to 0.8 mm with the first side wall, which will not protrude too much and occupy space, and at the same time can reduce the gap between the rotating table and the first side wall. Friction improves the smoothness of rotation.
  • the rotating base further includes a connecting base that passes through the first side wall along the extension direction of the rotating center line, and the rotating driving device is provided on the first side wall.
  • the rotating platform On one side of the first side wall, the rotating platform is provided on the other side of the first side wall, and a second limiting structure is provided between the main machine and the connecting base.
  • the second limiting structure is used to limit all The rotating platform moves toward the first side wall.
  • the second limiting structure can be used to maintain the above-mentioned gap and prevent the rotating table from moving in the axial direction during the rotation of the rotating base.
  • the connecting base has an annular convex surface, and the extension direction of the convex surface is perpendicular to the rotation centerline;
  • the second limiting structure includes a plurality of limiting members and the convex surface, a plurality of The limiters are arranged at intervals along the circumferential direction of the connecting seat.
  • the limiters and the rotating platform are respectively located on different sides of the first side wall.
  • Each of the limiters is connected to the first side wall.
  • the convex surfaces are in contact with each other.
  • the second limiting structure mainly uses a method of pushing the connecting seat outward from the inner side of the first side wall to prevent the rotating table from moving toward the inner side of the first side wall.
  • the connecting base has a first part and a second part that are connected along the extension direction of the rotation center line, and the outer diameter of the first part is larger than the outer diameter of the second part, so The side of the first part facing the second part is the convex surface, and each of the limiting members is relatively rotatable about its own axis and is disposed on the first side wall, and each of the limiting members is The outer peripheral surfaces of the positioning members all abut against the outer peripheral surface of the second part, and the limiting members are in rolling contact with the second part.
  • the limiter can limit the second part of the connecting seat in the circumferential direction, and the limiter contacts the second part by rolling friction, which helps to reduce the friction caused by the limiter and improve the smooth rotation of the rotating seat. sex.
  • the flow electroporation device further includes a limiting member that is connected to the host in a manner that can relatively rotate around its own axis, and a plurality of the limiting members are arranged at intervals;
  • the rotating base further includes a connecting base, and the limiting member abuts against the outer peripheral surface of the connecting base; further, in some embodiments, the limiting member and the connecting base The outer peripheral surface is in rolling contact.
  • a plurality of stoppers can be used to provide support and rotation guidance for the rotating base.
  • the main machine has a first side wall
  • the rotating base is relatively rotatably connected to the first side wall around the rotation center line
  • the limiting member is connected to the first side wall.
  • the walls are connected, and the connecting seat passes through the first side wall along the extension direction of the rotation center line.
  • Both the rotating seat and the limiting piece are connected to the first side wall, and the supporting and guiding functions of the limiting piece are more stable and reliable.
  • the flow electroporation device realizes switching between the first working state and the second working state through the rotation of the electrode assembly relative to the host machine, so that the position of the liquid outlet can be changed as needed. After the cell suspension is electroporated, part or all of the remaining cell suspension can be discharged from the electrode assembly by its own gravity alone, thereby improving the cell recovery rate.
  • FIG. 1 is a schematic front view of the flow electroporation device in a first working state in a specific embodiment of the present invention
  • Figure 2 is a schematic front view of the flow electroporation device in the second working state in this embodiment
  • Figure 3 is an exploded schematic diagram of the structure of the electrode assembly, rotating base and rotating motor in this embodiment
  • Figure 4 is a schematic perspective view of the assembly of the electrode assembly, rotating base and rotating motor in this embodiment. At this time, the flow electroporation device is in the first working state;
  • Figure 5 is a schematic perspective view of the assembly of the electrode assembly, rotating base and rotating motor in this embodiment. At this time, the flow electroporation device is in the second working state;
  • Figure 6 is a partial perspective view of the flow electroporation device in this embodiment.
  • Figure 7 is an exploded schematic diagram of the partial structure of the flow electroporation device in this embodiment.
  • Figure 8 is a schematic rear view of the rotating base and motor bracket in this embodiment
  • Electrode assembly 210 , liquid inlet; 220, liquid outlet; 230, shell; 310, sampling container; 311, first channel port; 312, liquid inlet pipeline; 320, collection container; 321, second channel port; 322, Liquid outlet pipeline; 400, liquid level sensor; 401, pipeline guide groove; 500, peristaltic pump; 600, rotating seat; 610, mounting groove; 620, rotating table; 630, connecting seat; 631, first part; 632. The second part; 633. Limiting member; 640. The main body of the rotating seat; 641.
  • the connecting part; 642. The convex part; 710. The rotating motor; 711. The main body of the motor; 712. The output shaft; 720.
  • a flow electroporation device is used for electroporation of cell suspension.
  • the flow electroporation device includes a host 100, an electrode assembly 200, a sampling container 310, a collection container 320, a liquid level sensor 400, a peristaltic pump 500, a rotating base 600, a rotational driving device, etc. provided on the host 100.
  • the host 100 is generally in the shape of a rectangular parallelepiped. Taking the direction shown in the figure as a reference, the host 100 has a first side wall 110 located on the front side, and a first side wall 110 located separately on the first side wall 110 .
  • the extension directions intersect.
  • first side wall 110 , the second side wall 120 , and the third side wall 130 respectively extend in the vertical direction, and the first side wall 110 is perpendicular to the second side wall 120 and the third side wall 130 respectively.
  • the side wall 120 and the third side wall 130 are parallel to each other.
  • the upper part of the first side wall 110 is the control panel 111 , and a rotation driving device and a control system are provided inside the host 100 .
  • the second side wall 120 is provided with a first bracket 121 for hanging the sampling container 310; the third side wall 130 is provided with a second bracket 131 for hanging the sampling container 310.
  • the first bracket 121 is located relatively above the second bracket 131. The cell suspension can be transferred from the sampling container 310 to the collection container 320, and the sampling container 310 and the collection container 320 are both located outside the corresponding side walls.
  • the electrode assembly 200 , the liquid level sensor 400 and the peristaltic pump 500 are all arranged outside the first side wall 110 .
  • the electrode assembly 200 includes a shell 230 with an inner cavity. The inner cavity is provided with electrodes for electroporation of cell suspensions (not shown in the figure).
  • the electrode assembly 200 also has a shell 230 provided on the shell 230
  • the liquid inlet 210 and the liquid outlet 220 are respectively located on different sides of the housing 230 .
  • the electrode assembly 200 is relatively rotatably disposed on one side of the host 100 , specifically, the electrode assembly 200 is relatively rotatably disposed on the first side wall 110 around the rotation center line 1001 .
  • the rotation center line 1001 is perpendicular to the first side wall 110 and extends in the horizontal direction.
  • the electrodes include a first electrode and a second electrode arranged at intervals. There is a processing channel between the first electrode and the second electrode for the cell suspension to flow. A strong electric field is formed between the first electrode and the second electrode, so that Electroporation can be performed when the cell suspension flows through the processing channel.
  • the sampling container 310 is used to store the cell suspension to be processed by the electrode.
  • the lower part of the sampling container 310 has a first channel opening 311.
  • the first channel opening 311 connects to the electrode assembly 200 through the liquid inlet pipe 312.
  • the liquid port 210 is connected.
  • the collection container 320 is used to receive the cell suspension treated by the electrode.
  • the upper part of the collection container 320 has a second channel opening 321 , and the second channel opening 321 is connected to the liquid outlet 220 of the electrode assembly 200 through a liquid outlet pipe 322 .
  • the liquid level sensor 400 and the peristaltic pump 500 are both fixed on the first side wall 110 , and the heights of the liquid level sensor 400 and the peristaltic pump 500 are both lower than the height of the electrode assembly 200 .
  • the liquid level sensor 400 is arranged between the sampling container 310 and the electrode assembly 200.
  • the liquid level sensor 400 has a pipeline guide groove 401.
  • the pipeline guide groove 401 extends in the vertical direction, and the liquid inlet pipeline 312 extends from the bottom to the bottom. and passes through the pipeline guide groove 401.
  • the liquid level sensor 400 is further connected with the electrical signal of the control system in the host 100, and can measure the liquid level state in the liquid inlet pipe 312 and feed the signal back to the control system, and the control system controls the working state of the electrode assembly 200.
  • the peristaltic pump 500 is disposed between the collection container 320 and the electrode assembly 200, and the liquid outlet pipeline 322 passes through the peristaltic pump 500 from top to bottom.
  • the rotating base 600 is disposed on one side of the host 100 so as to be relatively rotatable around the rotation centerline 1001 .
  • the rotational driving device is used to drive the rotating base 600 to rotate.
  • the electrode assembly 200 is detachably disposed on the rotating base. 600 and can rotate synchronously with the rotating base 600.
  • the flow electroporation device has a first working state and a second working state.
  • the electrodes in the electrode assembly 200 can electroporate the cell suspension.
  • the height of the liquid inlet 210 is lower than the liquid outlet.
  • the height of the port 220, and the axis line of the liquid inlet 210 and the axis line of the liquid outlet 220 both extend in the vertical direction, and both are located in the same vertical plane perpendicular to the first side wall 110.
  • the processing channel between the electrodes also extends in the up and down direction, and the cell suspension can flow through the processing channel from bottom to top and receive electric shock.
  • the electric shock in the electrode assembly 200 stops.
  • the height of the liquid outlet 220 decreases, so that the cell suspension in the electrode assembly 200 can It flows out from the liquid outlet 220 under the action of gravity.
  • the height of the liquid outlet 220 is lower than the height of the liquid inlet 210 in the second working state.
  • the liquid outlet 220 rotates about 150° clockwise around the rotation center line 1001 and then switches to the second working state.
  • the cross-sectional area of the processing channel is larger than the cross-sectional area of the outlet pipe 322
  • the liquid outlet 220 is lowered, so that under the combined action of gravity and the suction of the peristaltic pump 500, the cell suspension remaining in the treatment channel can be fully discharged. and recycled to collection container 320.
  • the peristaltic pump 500 when the flow electroporation device is in the first working state and the second working state, the peristaltic pump 500 always keeps working, so that it can cooperate with the gravity of the cell suspension itself to suspend the cells. Liquid continues to be fully drawn from the electrode assembly 200. In other embodiments, when the flow electroporation device is in the second working state, the peristaltic pump 500 can also stop working, and at this time, the cell suspension is discharged from the electrode assembly 200 only by its own gravity.
  • a new batch of cell suspension can be processed.
  • the electrode assembly 200 rotates counterclockwise about 150° around the rotation center line 1001 and then switches from the second working state to the first working state, so that a new round of electroporation of the cell suspension can be started.
  • the rotary drive device specifically adopts a rotary motor 710.
  • a motor bracket 720 is fixed on the host 100.
  • the motor bracket 720 is specifically fixed on the inside of the first side wall 110.
  • the motor The bracket 720 includes a support plate 721 extending in the up and down direction and a connecting plate 722 extending in the horizontal direction.
  • the upper part of the supporting plate 721 and the rear part of the connecting plate 722 are fixedly arranged.
  • the front part of the connecting plate 722 is fixed on the first side wall 110
  • On the support plate 721, a limiting hole 723 is formed.
  • the rotating electrical machine 710 includes a motor body 711 and an output shaft 712 .
  • the output shaft 712 is disposed on the motor body 711 so as to be relatively rotatable around the rotation center line 1001 .
  • the motor body 711 is fixed on one side of the support plate 721, and the rotating base 600 is relatively rotatably located on the other side of the supporting plate 721.
  • the rotating base 600 is located on the side close to the first side wall 110, and the motor body 711 is located on the other side of the supporting plate 721.
  • the output shaft 712 passes through the limiting hole 723 and is connected to the connecting portion 641 of the rotating base 600 , so that the output shaft 712 can drive the entire rotating base 600 to rotate.
  • the rotating base 600 mainly includes a rotating platform 620, a connecting base 630 and a rotating base main body 640 which are arranged in sequence along the extension direction of the rotating center line 1001. Different parts can be integrally formed or made separately and then fixed together. Among them, the main body 640 of the rotating seat is in the shape of a rectangular parallelepiped, the rotating platform 620 and the connecting seat 630 are both in the shape of a disc, and the outer diameter of the rotating platform 620 is larger than the outer diameter of the connecting seat 630.
  • the connecting seat 630 passes along the extension direction of the rotating center line 1001. Through the first side wall 110 , the rotating platform 620 is located outside the first side wall 110 , and the rotating base body 640 is located inside the first side wall 110 .
  • the rotating base 600 is provided with a mounting slot 610. The opening of the mounting slot 610 is located on the rotating platform 620.
  • the housing 230 of the electrode assembly 200 can be inserted into the mounting slot 610 in a form-fitting manner.
  • this embodiment provides a third limiting structure between the electrode assembly 200 and the rotating base 600.
  • the limitation is that the electrode assembly 200 can only be installed on the rotating base 600 in a preset direction.
  • the connecting seat 630 and the electrode assembly 200 cannot rotate 360° around the rotation center line 1001. Instead, there are a first limit position of rotation and a third limit position of the rotation. Two extreme positions. Specifically, first define the position of the electrode assembly 200 when the flow electroporation device is in the first working state as the reference position. There is a virtual baseline 1002.
  • the baseline 1002 is a ray perpendicular to the rotation center line 1001 and extending vertically upward.
  • the baseline The endpoint of 1002 is located on the rotation center line 1001, and the direction in which the electrode assembly 200 rotates from the reference position to the second limit position is the positive direction (clockwise rotation as shown in the figure is the positive direction).
  • the axis 1003 of the liquid outlet coincides with the baseline 1002; when the electrode assembly 200 is in the first extreme position, the angle between the axis 1003 of the liquid outlet and the baseline 1002 is -10°, that is, the liquid outlet 220 is slightly tilted to the upper left; when the electrode assembly 200 is at the second extreme position, the angle between the axis 1003 of the liquid outlet and the baseline 1002 is 190°, that is, the liquid outlet The mouth 220 is slightly inclined to the lower left.
  • a first limiting structure is provided between the rotating base 600 and the host 100 .
  • the first limiting structure is used to limit the electrode assembly 200 to only be able to operate in the above-mentioned first limit position and second limit position. Rotation between them means that it can only rotate forward from the first extreme position to the second extreme position, or reversely rotate from the second extreme position to the first extreme position.
  • the normal operation of the flow electroporation device mainly involves the aforementioned first working state and the second working state.
  • the electrode assembly 200 generally does not need to rotate to the first limit position or the second limit position, and the electrode assembly 200 does not need to rotate to the first limit position or the second limit position.
  • the specific tilt angle of the 200 within the rotatable range is also adjustable.
  • the first limiting structure includes a convex portion 642 and a limiting hole 723.
  • the tail part of the rotating base body 640 has the above-mentioned connecting part 641.
  • the rotating center line 1001 passes through the connecting part 641.
  • the convex part 642 is fixed on the connecting part 641 and protrudes outward along the radial direction of the rotating center line 1001.
  • the connecting part 641 and The protrusions 642 are inserted into the limiting holes 723 .
  • the side wall of the limiting hole 723 has a first boss 723a and a second boss 723b arranged at intervals, and the central angle between the first boss 723a and the second boss 723b is The angle is approximately 200°.
  • the protruding portion 642 When the electrode assembly 200 is in the first extreme position, the protruding portion 642 abuts on the first boss 723a; when the electrode assembly 200 is in the second extreme position, the protruding portion 642 abuts on the second boss 723b.
  • the rotation platform 620 is spaced apart from the first side wall 110 , and the liquid inlet 210 and the liquid outlet 220 are both exposed outside the installation groove 610 .
  • a gap of about 0.5 to 0.8 mm can be maintained between the rotating platform 620 and the first side wall 110 to avoid direct contact and friction between the two and affect the rotation.
  • a second limiting structure is provided between the host 100 and the connecting base 630 . The second limiting structure is used to limit the movement of the rotating platform 620 toward the first side wall 110 .
  • the connecting seat 630 has a first part 631 and a second part 632 that are connected along the extension direction of the rotation center line 1001.
  • the outer diameter of the first part 631 is larger than the outer diameter of the second part 632, so that the first part 631 faces
  • One side of the second part 632 has an annular convex surface, and the extension direction of the convex surface is perpendicular to the rotation center line 1001.
  • the outer peripheral surface of the second part 632 is specifically a cylindrical surface.
  • the second limiting structure includes a limiting member 633.
  • the limiting member 633 has a plurality of limiting members 633 arranged at intervals along the circumferential direction of the second part 632.
  • the limiting member 633 is specifically a roller.
  • Each limiting member 633 can rotate around its own axis.
  • the wires are relatively rotatably arranged on the first side wall 110, and the limiting member 633
  • the axis line is parallel to the rotation center line 1001
  • the limiting member 633 and the rotation driving device are located on the same side of the first side wall 110, that is, located inside the first side wall 110.
  • One axial side of each limiting member 633 is against the annular convex surface, and the outer peripheral surface of each limiting member 633 is against the outer peripheral surface of the second portion 632 .
  • the limiting member 633 of the roller is in rolling contact with the second portion 632 .
  • limiting members 633 evenly spaced along the circumferential direction of the second part 632, which not only limit the axial movement of the rotating stage 620, but also provide rotation guidance for the limiting members 633, and
  • the rotating base 600 is auxiliary supported, which reduces the load of the rotating motor 710 to a certain extent.
  • the device in order to avoid insufficient torque of the rotating motor 710 resulting in poor rotation or noise, the device is designed to be lightweight and requires that the load of the rotating motor 710 should not exceed 1kg.
  • the rotating base 600 is made of lightweight materials and weighs about 0.4kg.
  • the actual load of the rotating motor 710 is lower, so that it can operate stably.
  • the flow electroporation device provided in this embodiment can not only significantly improve the uniformity and continuity of the cell suspension flow during the electroporation process, improve the electroporation efficiency, but also can process the cell suspension after treatment. Fully discharged, improved cell recovery rate, and the flow electroporation device operates stably, reliably, and is easy to operate.

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Abstract

提供了一种流式电穿孔装置，包括主机及设于其上的电极组件、进样容器及收集容器。电极组件包括用于对细胞悬液进行电穿孔处理的电极，电极组件具有进液口与出液口，电极组件能够相对转动地设置在主机的一侧；进样容器用于存放待电极处理的细胞悬液，进样容器与进液口相连通；收集容器用于接收电极处理过的细胞悬液，收集容器与出液口相连通。该流式电穿孔装置具有第一工作状态与第二工作状态，第一工作状态下，电极对细胞悬液进行电穿孔处理；电极组件相对主机旋转而转换为第二工作状态；第二工作状态下，电极组件中的至少部分细胞悬液能够在重力作用下从出液口流出。该装置能够在细胞处理时减少气泡产生、提高细胞处理效率，在处理过后提高细胞回收率。

Description

一种流式电穿孔装置 技术领域
本发明涉及生物技术领域，尤其涉及一种流式电穿孔装置。
背景技术
电穿孔(Electroporation)是一种利用电场作用在细胞膜上，使细胞膜产生可供DNA、RNA、蛋白质等外源分子通过的微孔的技术，这些微孔在电场作用下的瞬间产生，并可随电场的消失而恢复，从而避免对细胞的永久性伤害。利用电穿孔技术可以将DNA、RNA、蛋白质、糖类、染料、病毒颗粒等多种外源分子导入原核或真核细胞内，称为电转染技术(Electrotransfection)，电转染技术在抗体蛋白生产、体外诊断(IVD)试剂原料生产、免疫细胞的基因编辑和基因修饰等诸多高端生物医药技术领域均有广泛的应用，是对细胞内进行物质递送的重要技术。
对细胞进行电穿孔处理的方法有多种，其中，流式电穿孔技术能够对流动的含有细胞的悬浮液(以下简称细胞悬液)进行持续的电穿孔处理，较为高效，尤其适用于大体积细胞悬液的批量处理。常见的流式电穿孔装置主要包括一对用于产生电场的平面电极，以及位于两个平面电极之间的腔室，细胞悬液流经腔室的同时能够受到电击，产生电穿孔，从而细胞悬液中的外源分子能够进入目标细胞中。现有技术中，流式电穿孔装置大多存在细胞处理效率偏低、细胞悬液残留量较大等问题。一方面，细胞悬液接受电击时会产生水电解现象，容易产生大量气泡，导致液流不连续或不均匀、流速不稳定等问题，降低了电场均匀性，同时影响了细胞接受电击次数的可控性，从而导致细胞转染效率的下降；另一方面，电穿孔过程中产生的气泡会形成泡沫，其与细胞混合容易导致细胞悬液滞留在腔室和管路中，难以排尽，降低了流式电穿孔装置最终的细胞回收率。
发明内容
本发明的目的是针对现有技术存在的问题，提供一种细胞处理效率高的流式电穿孔装置。
为达到上述目的，本发明采用的技术方案是：
一种流式电穿孔装置，所述流式电穿孔装置包括主机、电极组件、进样容器及收集容器，所述电极组件、所述进样容器及所述收集容器分别设于所述主机上，其中，
所述电极组件包括用于对细胞悬液进行电穿孔处理的电极，所述电极组件具有进液口与出液口，所述电极组件能够相对转动地设置在所述主机的一侧；
所述进样容器用于存放待所述电极处理的细胞悬液，所述进样容器通过进液管路与所述进液口相连通；
所述收集容器用于接收所述电极处理过的细胞悬液，所述收集容器通过出液管路与所述出液口相连通；
所述流式电穿孔装置具有第一工作状态与第二工作状态，当所述流式电穿孔装置处于所述第一工作状态下，所述电极对细胞悬液进行电穿孔处理；当所述流式电穿孔装置由所述第一工作状态向所述第二工作状态转换的过程中，所述电极组件相对所述主机旋转；当所述流式电穿孔装置处于所述第二工作状态下，所述电极组件中的至少部分细胞悬液能够在重力作用下从所述出液口流出。
在一些实施方式中，当所述流式电穿孔装置处于所述第一工作状态下，所述进液口的高度低于所述出液口的高度；当所述流式电穿孔装置处于所述第二工作状态下，所述出液口的高度降低。当第一工作状态下，流式电穿孔装置能够正常运行，细胞悬液以下进上出的流向持续流经电极组件并进行电穿孔处理，在细胞悬液自身重力的作用下能够充盈电极片之间的腔室，保持电极片之间电联通，提高了液流的连续性与均匀性，有效减少气泡产生，又提高了电场的均匀性，从而综合提升了电穿孔效率。当第二工作状态下，出液口的高度降低，使得腔室内未排尽的细胞悬液能够在自身重力作用下排出。
在一些实施方式中，当所述流式电穿孔装置处于所述第二工作状态下，所述出液口的高度低于所述进液口的高度。从而电极组件具有更大的倾斜角度，有助于细胞悬液更加充分地排出。
在一些实施方式中，当所述流式电穿孔装置处于所述第一工作状态下，所述进液口的轴心线及所述出液口的轴心线均沿竖直方向延伸。从而当第一工作状态下，细胞悬液竖直向上地流经电极组件，能够更好地保障液流的均匀性和电场均匀性。
在一些实施方式中，所述电极包括间隔设置的第一电极与第二电极，所述第一电极与所述第二电极之间具有处理通道，所述流式电穿孔装置处于所述第一工作状态下，所述处理通道沿上下方向延伸，所述细胞悬液自下而上流经所述处理通道。从而细胞悬液不仅在电极组件整体的进液口与出液口中下进上出，且在具体的电极间的处理通道中也能够保持下进上出的流向，进一步确保电极之间腔室中液流的均匀性和电场均匀性。
在一些实施方式中，所述流式电穿孔装置还包括设置在所述进样容器和所述电极组件之间的液位传感器，所述液位传感器固设于所述主机的一侧，所述液位传感器的高度低于所述电极组件的高度，所述液位传感器具有管路导引槽，能够与进液管路紧密贴合，有利于液位传感器正常工作。所述管路导引槽沿竖直方向延伸，所述进液管路自下而上穿过所述管路导引槽。液位传感器可用于测量进液管路中的液位状态并将信号传输给控制系统，由控制系统控制电极组件的工作状态。液位传感器采用竖直放置且进样管路中细胞悬液下进上出的工作 方式，可确保管路导引槽中的进液管路内充满液体，避免管路内液体不满或气泡对检测结果造成误判，影响控制系统对电穿孔过程的正确控制。
在一些实施方式中，所述流式电穿孔装置还包括蠕动泵，所述蠕动泵固设于所述主机的一侧，所述出液管路自上而下穿过所述蠕动泵。蠕动泵用于提供细胞悬液在整个流式电穿孔装置中流动的动力，此处将蠕动泵设置在收集容器和电极组件之间，安装于电极组件的后端及下方，蠕动泵与电极组件的出液管路结合更为顺畅。当蠕动泵启动时，在电极组件及其前侧的管路内形成的负压，将进样容器中细胞悬液抽吸流出；当整个管路充满液体时，管路内部压力达到平衡，蠕动泵稳定传输细胞悬液。相较于蠕动泵安装在进液管路上而在电极组件及其后侧的管路内形成正压、推动细胞悬液流动，本发明中细胞悬液的流速更加均匀稳定，有助于获得更好的电穿孔处理效果。
在一些实施方式中，所述流式电穿孔装置还包括液位传感器及蠕动泵，所述主机具有第一侧壁，以及分设于所述第一侧壁宽度方向相异两侧的第二侧壁与第三侧壁，所述第一侧壁的延伸方向与所述第二侧壁的延伸方向相交，且所述第一侧壁的延伸方向与所述第三侧壁的延伸方向相交；所述电极组件能够相对转动地设置在所述第一侧壁上，所述液位传感器、所述蠕动泵均设置在所述第一侧壁上；所述进样容器设于所述第二侧壁上，所述收集容器设于所述第三侧壁上。
在一些具体实施方式中，第一侧壁与第二侧壁垂直，且第一侧壁与第三侧壁也垂直。从而优化了该流式电穿孔装置的空间布局，液位传感器、电极组件、蠕动泵设置在同一侧壁上，管路排布更为整齐，电极组件的旋转不易导致管路交叉缠绕，确保液流通畅；进样容器与收集容器分设于左右两侧壁(即第二侧壁与第三侧壁)，位置较为固定，不易受到电极组件旋转的影响，且有助于缩短主机的宽度，减小主机占用空间。
在一些实施方式中，所述流式电穿孔装置还包括转动座及旋转驱动装置，所述转动座能够绕转动中心线相对转动地与所述主机相连接，所述旋转驱动装置用于驱动所述转动座旋转，所述电极组件可拆卸地设置在所述转动座上并能够与所述转动座同步旋转。转动座作为中间部件，有助于提高电极组件转动时的稳定性与可靠性，电极组件与转动座同步运动。
在一些实施方式中，所述转动中心线沿水平方向延伸，存在一条基线，所述基线为垂直于所述转动中心线且竖直向上延伸的射线，所述基线的端点位于所述转动中心线上，所述电极组件绕所述转动中心线转动的过程中具有第一极限位置、第二极限位置及基准位置，所述电极组件由所述基准位置向所述第二极限位置转动的方向为正方向；当所述电极组件处于所述基准位置时，所述出液口的轴心线与所述基线相重合；当所述电极组件处于所述第一极限位置时，所述出液口的轴心线与所述基线之间的夹角为-10°；当所述电极组件处于所述第二 极限位置时，所述出液口的轴心线与所述基线之间的夹角为190°。
在一些实施方式中，所述转动座与所述主机之间设有第一限位结构，所述第一限位结构用于限制所述电极组件在所述第一极限位置与所述第二极限位置之间转动。第一限位结构对电极组件的旋转角度进行了限定，电极组件只能够在预设的极限位置之间转动。一般地，第一工作状态下，电极组件处于基准位置；第二工作状态下，出液口的轴心线与基线之间的夹角约为150°，此时电极组件出液口附近管路中残留的细胞悬液就能够较好地排尽。第一限位结构有效避免了过大旋转角度可能导致的管路拉扯或打结问题，提高该流式电穿孔装置运行的可靠性。
在一些实施方式中，所述主机设有沿上下方向延伸的支撑板，所述支撑板上开设有限位孔；所述转动座具有连接部，所述转动中心线穿过所述连接部，所述连接部具有沿所述转动中心线的径向向外凸出的凸部，所述连接部插设在所述限位孔中；所述第一限位结构包括所述凸部及所述限位孔，沿所述转动中心线的周向，所述限位孔的侧壁具有间隔设置的第一凸台与第二凸台；当所述电极组件处于所述第一极限位置时，所述凸部与所述第一凸台相抵靠；当所述电极组件处于所述第二极限位置时，所述凸部与所述第二凸台相抵靠。第一限位结构具体采用简单的凸部与两组凸台相配合的结构，设计简单、不占空间、可靠性强。
在一些实施方式中，所述主机具有第一侧壁，所述转动座能够绕所述转动中心线相对转动地与所述第一侧壁相连接，所述转动中心线垂直于所述第一侧壁，所述转动座包括转动台，沿所述转动中心线的延伸方向，所述转动台与所述第一侧壁之间设有间隙，所述进液口与所述出液口均位于所述转动台的远离所述第一侧壁的一侧。转动台为电极组件的转动提供支撑平面，优选转动台与第一侧壁保持约0.5～0.8mm的运动间隙，既不会过于凸出、占用空间，同时可降低与第一侧壁之间的摩擦，提高转动的流畅性。
在一些实施方式中，所述转动座还包括连接座，所述连接座沿所述转动中心线的延伸方向穿过所述第一侧壁，所述旋转驱动装置设于所述第一侧壁的一侧，所述转动台设于所述第一侧壁的另一侧，所述主机与所述连接座之间设有第二限位结构，所述第二限位结构用于限制所述转动台朝向所述第一侧壁运动。第二限位结构可用于保持上述间隙，避免转动座转动过程中转动台沿轴向运动。
在一些实施方式中，所述连接座具有环状的凸面，所述凸面的延伸方向垂直于所述转动中心线；所述第二限位结构包括多个限位件及所述凸面，多个所述限位件沿所述连接座的周向间隔设置，所述限位件与所述转动台分设于所述第一侧壁的相异两侧，每个所述限位件均与所述凸面相抵靠。第二限位结构主要采用从第一侧壁内侧向外顶推连接座的方式防止转动台向第一侧壁内侧移动。
在一些实施方式中，所述连接座具有沿所述转动中心线的延伸方向相接的第一部与第二部，所述第一部的外径大于所述第二部的外径，所述第一部朝向所述第二部的一侧为所述凸面，每个所述限位件均能够绕自身轴心线相对旋转地设置在所述第一侧壁上，每个所述限位件的外周面均抵靠在所述第二部的外周面上，所述限位件与所述第二部滚动接触。限位件能够在周向上对连接座的第二部进行限位，并且限位件采用滚动摩擦的方式与第二部接触，有助于减小限位造成的摩擦，提高转动座转动的流畅性。
在一些实施方式中，所述流式电穿孔装置还包括能够绕自身轴心线相对旋转地与所述主机相连的限位件，多个所述限位件间隔设置；
在一些实施方式中，所述转动座还包括连接座，所述限位件与所述连接座的外周面相抵靠；进一步地，在一些实施例中，所述限位件与所述连接座的外周面滚动接触。多个限位件可用于提供转动座的支撑与旋转导向。
在一些实施方式中，所述主机具有第一侧壁，所述转动座能够绕所述转动中心线相对转动地与所述第一侧壁相连接，所述限位件与所述第一侧壁相连，所述连接座沿所述转动中心线的延伸方向穿过所述第一侧壁。转动座与限位件均与第一侧壁相连，限位件的支撑及导向作用更为稳定可靠。
由于上述技术方案的运用，本发明提供的流式电穿孔装置，通过电极组件相对于主机的转动来实现第一工作状态与第二工作状态之间的切换，从而可以根据需要改变出液口的朝向，在细胞悬液被电穿孔处理过后，其残留的部分或全部细胞悬液可以仅靠自身重力排出电极组件，提高细胞回收率。
附图说明
为了更清楚地说明本发明的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。
附图1为本发明一具体实施例中流式电穿孔装置处于第一工作状态下的主视示意图；
附图2为本实施例中流式电穿孔装置处于第二工作状态下的主视示意图；
附图3为本实施例中电极组件、转动座及旋转电机的结构分解示意图；
附图4为本实施例中电极组件、转动座及旋转电机的装配立体示意图，此时流式电穿孔装置处于第一工作状态；
附图5为本实施例中电极组件、转动座及旋转电机的装配立体示意图，此时流式电穿孔装置处于第二工作状态；
附图6为本实施例中流式电穿孔装置的局部立体示意图；
附图7为本实施例中流式电穿孔装置的局部结构分解示意图；
附图8为本实施例中转动座与电机支架的后视示意图；
其中：100、主机；110、第一侧壁；111、控制面板；120、第二侧壁；121、第一支架；130、第三侧壁；131、第二支架；200、电极组件；210、进液口；220、出液口；230、壳体；310、进样容器；311、第一通道口；312、进液管路；320、收集容器；321、第二通道口；322、出液管路；400、液位传感器；401、管路导引槽；500、蠕动泵；600、转动座；610、安装槽；620、转动台；630、连接座；631、第一部；632、第二部；633、限位件；640、转动座主体；641、连接部；642、凸部；710、旋转电机；711、电机主体；712、输出轴；720、电机支架；721、支撑板；722、连接板；723、限位孔；723a、第一凸台；723b、第二凸台；1001、转动中心线；1002、基线；1003、出液口的轴心线。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域的技术人员理解。
参见图1至图8所示，一种流式电穿孔装置，用于对细胞悬液进行电穿孔处理。该流式电穿孔装置包括主机100，以及设于主机100上的电极组件200、进样容器310、收集容器320、液位传感器400、蠕动泵500、转动座600及旋转驱动装置等。
参见图1及图2所示，本实施例中，主机100大体呈长方体状，以图示的方向为参考，主机100具有位于前侧的第一侧壁110，以及分设于第一侧壁110左右两侧的第二侧壁120与第三侧壁130，第一侧壁110的延伸方向与第二侧壁120的延伸方向相交，且第一侧壁110的延伸方向与第三侧壁130的延伸方向相交。实际上，第一侧壁110、第二侧壁120、第三侧壁130分别沿竖直方向延伸，且第一侧壁110与第二侧壁120、第三侧壁130分别垂直，第二侧壁120与第三侧壁130相互平行，第一侧壁110的上部为控制面板111，主机100的内部设有旋转驱动装置及控制系统等。
本实施例中，第二侧壁120上设有第一支架121，第一支架121用于悬挂进样容器310；第三侧壁130上设有第二支架131，第二支架131用于悬挂收集容器320，第一支架121相对位于第二支架131的上方，细胞悬液能够从进样容器310向收集容器320传输，且进样容器310与收集容器320均设于对应侧壁的外侧。
本实施例中，电极组件200、液位传感器400及蠕动泵500均设置在第一侧壁110的外侧。其中，电极组件200包括具有内腔的壳体230，内腔中设有用于对细胞悬液进行电穿孔处理的电极(图中未示出)，电极组件200还具有分别开设在壳体230上的进液口210与出液口220，进液口210与出液口220分设于壳体230的相异两侧。电极组件200能够相对转动地设置在主机100的一侧，具体是能够绕转动中心线1001相对转动地设置在第一侧壁110 上，转动中心线1001垂直于第一侧壁110且沿水平方向延伸。进一步地，电极包括间隔设置的第一电极与第二电极，第一电极与第二电极之间具有可供细胞悬液流通的处理通道，第一电极与第二电极之间形成强电场，从而当细胞悬液流经处理通道时就能够进行电穿孔处理。
本实施例中，进样容器310用于存放待电极处理的细胞悬液，进样容器310的下部具有第一通道口311，第一通道口311通过进液管路312与电极组件200的进液口210相连通。收集容器320用于接收电极处理过的细胞悬液，收集容器320的上部具有第二通道口321，第二通道口321通过出液管路322与电极组件200的出液口220相连通。
本实施例中，液位传感器400及蠕动泵500均固设于第一侧壁110上，且液位传感器400与蠕动泵500的高度均低于电极组件200的高度。其中，液位传感器400设置在进样容器310和电极组件200之间，液位传感器400具有管路导引槽401，管路导引槽401沿竖直方向延伸，进液管路312自下而上穿过管路导引槽401。液位传感器400进一步与主机100内的控制系统电信号连接，可以测量进液管路312中的液位状态并将信号反馈给控制系统，由控制系统控制电极组件200的工作状态。蠕动泵500设置在收集容器320和电极组件200之间，出液管路322自上而下穿过蠕动泵500。
参见图3至图5所示，转动座600能够绕转动中心线1001相对转动地设置在主机100的一侧，旋转驱动装置用于驱动转动座600旋转，电极组件200可拆卸地设置在转动座600上并能够与转动座600同步旋转。
在旋转驱动装置的驱动下，该流式电穿孔装置具有第一工作状态与第二工作状态。参见图1及图4所示，当流式电穿孔装置处于第一工作状态下，电极组件200中的电极可对细胞悬液进行电穿孔处理，此时进液口210的高度低于出液口220的高度，且进液口210的轴心线及出液口220的轴心线均沿竖直方向延伸，两者位于同一个垂直于第一侧壁110的竖直平面内，此时电极间的处理通道也沿上下方向延伸，细胞悬液能够自下而上流经处理通道并接受电击。如此可确保整个处理通道被细胞悬液充满，且细胞悬液流速均匀，同时电穿孔过程中产生的气泡可以快速通过壳体230顶部的出液口220排出，不会在处理通道内部滞留而影响电穿孔效果。
参见图2及图5所示，当流式电穿孔装置处于第二工作状态下，电极组件200中的电击停止，此时出液口220的高度降低，使得电极组件200中的细胞悬液能够在重力作用下从出液口220流出，本实施例中第二工作状态下出液口220的高度低于进液口210的高度。具体地，与第一工作状态相比，出液口220绕转动中心线1001顺时针旋转约150°后转换为第二工作状态。由于处理通道的横截面积大于出液管路322的横截面积，电穿孔处理后，由于细胞悬液表面张力小及细胞悬液自重的作用，处理通道内会残留一部分细胞悬液无法排出，造 成细胞损失。此时通过旋转驱动装置带动转动座600及电极组件200旋转一定角度后，出液口220降低，从而在重力和蠕动泵500吸力的共同作用下，处理通道内滞留的细胞悬液得以充分排出，并回收到收集容器320。
需要说明的是，本实施例中，当流式电穿孔装置处于第一工作状态与第二工作状态下，蠕动泵500始终保持工作，从而能够与细胞悬液自身的重力相配合，将细胞悬液持续地从电极组件200中充分吸出。在其他实施例中，当流式电穿孔装置处于第二工作状态下，蠕动泵500也可以暂停工作，此时细胞悬液仅依靠自身重力从电极组件200中排出。
当一批细胞悬液处理结束后，可进行新的一批细胞悬液处理。在旋转驱动装置的驱动下，电极组件200绕转动中心线1001逆时针旋转约150°后由第二工作状态转换为第一工作状态，从而可以开始新一轮对细胞悬液的电穿孔处理。
参见图6及图7所示，本实施例中，旋转驱动装置具体采用旋转电机710，主机100上固设有电机支架720，电机支架720具体是固设于第一侧壁110的内侧，电机支架720包括沿上下方向延伸的支撑板721及沿水平方向延伸的连接板722，支撑板721的上部与连接板722的后部固定设置，连接板722的前部固设于第一侧壁110上，支撑板721上开设有限位孔723。旋转电机710包括电机主体711与输出轴712，输出轴712能够绕转动中心线1001相对旋转地设置于电机主体711上。电机主体711固设于支撑板721的一侧，转动座600能够相对转动地设于支撑板721的另一侧，具体是转动座600位于靠近第一侧壁110的一侧，电机主体711位于远离第一侧壁110的一侧，输出轴712穿设在限位孔723中并与转动座600的连接部641相连接，进而输出轴712能够驱动整个转动座600旋转。
转动座600主要包括沿转动中心线1001的延伸方向依次设置的转动台620、连接座630及转动座主体640，不同部分可以是一体成型或分体制作后固定在一起。其中，转动座主体640呈长方体状，转动台620与连接座630均呈圆盘状，且转动台620的外径大于连接座630的外径，连接座630沿转动中心线1001的延伸方向穿过第一侧壁110，转动台620位于第一侧壁110的外侧，转动座主体640位于第一侧壁110的内侧。转动座600上开设有安装槽610，安装槽610的开口位于转动台620上，电极组件200的壳体230能够形状配合地插设在安装槽610中。
参见图3所示，为了确保细胞悬液只能够以预设的方向流入电极组件200，保证细胞处理效果，本实施例在电极组件200与转动座600之间设置了第三限位结构，用于限制电极组件200仅能够以预设方向安装到转动座600上。
参见图4及图5所示，为了提高装置的可靠性，本实施例中连接座630及电极组件200并不能绕转动中心线1001进行360°旋转，而是存在转动的第一极限位置与第二极限位置。 具体地，首先定义流式电穿孔装置处于第一工作状态时电极组件200的位置为基准位置，存在一条虚拟的基线1002，基线1002为垂直于转动中心线1001且竖直向上延伸的射线，基线1002的端点位于转动中心线1001上，以电极组件200由基准位置向第二极限位置转动的方向为正方向(图中所示顺时针旋转为正方向)。当电极组件200处于基准位置时，出液口的轴心线1003与基线1002相重合；当电极组件200处于第一极限位置时，出液口的轴心线1003与基线1002之间的夹角为-10°，即出液口220稍向左上方倾斜；当电极组件200处于第二极限位置时，出液口的轴心线1003与基线1002之间的夹角为190°，即出液口220稍向左下方倾斜。
参见图7及图8所示，转动座600与主机100之间设有第一限位结构，第一限位结构用于限制电极组件200仅能够在上述在第一极限位置与第二极限位置之间转动，即仅能够由第一极限位置正向旋转至第二极限位置，或由第二极限位置反向旋转至第一极限位置。需要说明的是，该流式电穿孔装置正常运行过程中主要涉及前述的第一工作状态与第二工作状态，电极组件200一般不需要转动到第一极限位置或第二极限位置，且电极组件200在可转范围内的具体倾斜角度也是可调的。
本实施例中，第一限位结构包括凸部642及限位孔723。转动座主体640的尾部具有上述连接部641，转动中心线1001穿过连接部641，凸部642固设于连接部641上且沿转动中心线1001的径向向外凸出，连接部641及凸部642均插设在限位孔723中。沿转动中心线1001的周向，限位孔723的侧壁具有间隔设置的第一凸台723a与第二凸台723b，且第一凸台723a与第二凸台723b之间的圆心角夹角约为200°。当电极组件200处于第一极限位置时，凸部642抵靠在第一凸台723a上；当电极组件200处于第二极限位置时，凸部642抵靠在第二凸台723b上。
参见图6所示，本实施例中，沿转动中心线1001的延伸方向，转动台620与第一侧壁110间隔设置，进液口210与出液口220均露出于安装槽610之外并位于转动台620的远离第一侧壁110的一侧。转动台620与第一侧壁110之间具体能够保持约0.5～0.8mm的间隙，避免两者直接接触摩擦，影响转动。为了保持上述间隙，主机100与连接座630之间设有第二限位结构，第二限位结构用于限制转动台620朝向第一侧壁110运动。具体地，连接座630具有沿转动中心线1001的延伸方向相接的第一部631与第二部632，第一部631的外径大于第二部632的外径，从而第一部631朝向第二部632的一侧具有环状的凸面，凸面的延伸方向垂直于转动中心线1001，本实施例中，第二部632的外周面具体为一圆柱面。第二限位结构包括限位件633，限位件633具有沿第二部632的周向间隔设置的多个，限位件633具体为滚轮，每个限位件633均能够绕自身轴心线相对旋转地设置在第一侧壁110上，限位件633 的轴心线平行于转动中心线1001，且限位件633与旋转驱动装置位于第一侧壁110的同一侧，即位于第一侧壁110的内侧。每个限位件633的轴向的一侧面抵靠在上述环形的凸面上，且每个限位件633的外周面均抵靠在第二部632的外周面上，且本实施例中作为滚轮的限位件633与第二部632滚动接触。本实施例中，限位件633具有沿第二部632的周向均匀间隔设置的六个，既起到了限制转动台620轴向运动的作用，又能够为限位件633提供旋转导向，且对转动座600进行了辅助支撑，一定程度上降低了旋转电机710的载荷。
本实施例中，为了避免旋转电机710扭力不够导致转动不畅或产生噪声，对装置进行了轻量化设计，要求旋转电机710负载应不超过1kg。具体地，转动座600采用轻质材料制成，重量约0.4kg，同时由于上述限位件633承受了部分转动座600的重量，旋转电机710的实际承受的负载更低，从而能够稳定运行。
综上所述，本实施例提供的流式电穿孔装置，既能够在电穿孔过程中显著提高细胞悬液流动的均匀性与连续性，提高电穿孔效率，又能够在处理过后将细胞悬液充分排出，提高细胞回收率，且该流式电穿孔装置运行稳定可靠，易于操作。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (20)

  1. 一种流式电穿孔装置，其特征在于：所述流式电穿孔装置包括主机、电极组件、进样容器及收集容器，所述电极组件、所述进样容器及所述收集容器分别设于所述主机上，其中，
    所述电极组件包括用于对细胞悬液进行电穿孔处理的电极，所述电极组件具有进液口与出液口，所述电极组件能够相对转动地设置在所述主机的一侧；
    所述进样容器用于存放待所述电极处理的细胞悬液，所述进样容器通过进液管路与所述进液口相连通；
    所述收集容器用于接收所述电极处理过的细胞悬液，所述收集容器通过出液管路与所述出液口相连通；
    所述流式电穿孔装置具有第一工作状态与第二工作状态，当所述流式电穿孔装置处于所述第一工作状态下，所述电极对细胞悬液进行电穿孔处理；当所述流式电穿孔装置由所述第一工作状态向所述第二工作状态转换的过程中，所述电极组件相对所述主机旋转；当所述流式电穿孔装置处于所述第二工作状态下，所述电极组件中的至少部分细胞悬液能够在重力作用下从所述出液口流出。
  2. 根据权利要求1所述的流式电穿孔装置，其特征在于：当所述流式电穿孔装置处于所述第一工作状态下，所述进液口的高度低于所述出液口的高度；当所述流式电穿孔装置处于所述第二工作状态下，所述出液口的高度降低。
  3. 根据权利要求2所述的流式电穿孔装置，其特征在于：当所述流式电穿孔装置处于所述第二工作状态下，所述出液口的高度低于所述进液口的高度。
  4. 根据权利要求2所述的流式电穿孔装置，其特征在于：当所述流式电穿孔装置处于所述第一工作状态下，所述进液口的轴心线及所述出液口的轴心线均沿竖直方向延伸。
  5. 根据权利要求1-4任一权利要求所述的流式电穿孔装置，其特征在于：所述电极包括间隔设置的第一电极与第二电极，所述第一电极与所述第二电极之间具有处理通道，所述流式电穿孔装置处于所述第一工作状态下，所述处理通道沿上下方向延伸，所述细胞悬液自下而上流经所述处理通道。
  6. 根据权利要求1-5任一权利要求所述的流式电穿孔装置，其特征在于：所述流式电穿孔装置还包括设置在所述进样容器和所述电极组件之间的液位传感器，所述液位传感器固设于所述主机的一侧，所述液位传感器具有管路导引槽，所述管路导引槽沿竖直方向延伸，所述进液管路自下而上穿过所述管路导引槽。
  7. 根据权利要求1-6任一权利要求所述的流式电穿孔装置，其特征在于：所述流式电穿孔装置还包括蠕动泵，所述蠕动泵固设于所述主机的一侧，所述出液管路自上而下穿过所述蠕动泵。
  8. 根据权利要求1-7任一权利要求所述的流式电穿孔装置，其特征在于：所述流式电穿孔装置还包括液位传感器及蠕动泵，所述主机具有第一侧壁，以及分设于所述第一侧壁宽度方向相异两侧的第二侧壁与第三侧壁，所述第一侧壁的延伸方向与所述第二侧壁的延伸方向相交，且所述第一侧壁的延伸方向与所述第三侧壁的延伸方向相交；
    所述电极组件与所述第一侧壁能够相对转动地连接，所述液位传感器、所述蠕动泵均设置在所述第一侧壁上；所述进样容器设于所述第二侧壁上，所述收集容器设于所述第三侧壁上。
  9. 根据权利要求1-8任一权利要求所述的流式电穿孔装置，其特征在于：所述流式电穿孔装置还包括转动座及旋转驱动装置，所述转动座能够绕转动中心线相对转动地与所述主机相连接，所述旋转驱动装置用于驱动所述转动座旋转，所述电极组件可拆卸地设置在所述转动座上并能够与所述转动座同步旋转。
  10. 根据权利要求9所述的流式电穿孔装置，其特征在于：所述转动中心线沿水平方向延伸，存在一条基线，所述基线为垂直于所述转动中心线且竖直向上延伸的射线，所述基线的端点位于所述转动中心线上，所述电极组件绕所述转动中心线转动的过程中具有第一极限位置、第二极限位置及基准位置，所述电极组件由所述基准位置向所述第二极限位置转动的方向为正方向；
    当所述电极组件处于所述基准位置时，所述出液口的轴心线与所述基线相重合；
    当所述电极组件处于所述第一极限位置时，所述出液口的轴心线与所述基线之间的夹角为-10°；
    当所述电极组件处于所述第二极限位置时，所述出液口的轴心线与所述基线之间的夹角为190°。
  11. 根据权利要求10所述的流式电穿孔装置，其特征在于：所述转动座与所述主机之间设有第一限位结构，所述第一限位结构用于限制所述电极组件在所述第一极限位置与所述第二极限位置之间转动。
  12. 根据权利要求11所述的流式电穿孔装置，其特征在于：所述主机上设有沿上下方向延伸的支撑板，所述支撑板上开设有限位孔；所述转动座具有连接部，所述转动中心线穿过所述连接部，所述连接部具有沿所述转动中心线的径向向外凸出的凸部，所述连接部插设在所述限位孔中；所述第一限位结构包括所述凸部及所述限位孔，沿所述转动中心线的周向，所述限位孔的侧壁具有间隔设置的第一凸台与第二凸台；
    当所述电极组件处于所述第一极限位置时，所述凸部与所述第一凸台相抵靠；
    当所述电极组件处于所述第二极限位置时，所述凸部与所述第二凸台相抵靠。
  13. 根据权利要求9-12任一权利要求所述的流式电穿孔装置，其特征在于：所述主机具有第一侧壁，所述转动座能够绕所述转动中心线相对转动地与所述第一侧壁相连接，所述转动中心线垂直于所述第一侧壁，所述转动座包括转动台，沿所述转动中心线的延伸方向，所述转动台与所述第一侧壁之间设有间隙，所述进液口与所述出液口均位于所述转动台的远离所述第一侧壁的一侧。
  14. 根据权利要求13所述的流式电穿孔装置，其特征在于：所述转动座还包括连接座，所述连接座沿所述转动中心线的延伸方向穿过所述第一侧壁，所述旋转驱动装置设于所述第一侧壁的一侧，所述转动台设于所述第一侧壁的另一侧，所述主机与所述连接座之间设有第二限位结构，所述第二限位结构用于限制所述转动台朝向所述第一侧壁运动。
  15. 根据权利要求14所述的流式电穿孔装置，其特征在于：所述连接座具有环状的凸面，所述凸面的延伸方向垂直于所述转动中心线；所述第二限位结构包括多个限位件及所述凸面，多个所述限位件沿所述连接座的周向间隔设置，所述限位件与所述转动台分设于所述第一侧壁的相异两侧，每个所述限位件均与所述凸面相抵靠。
  16. 根据权利要求15所述的流式电穿孔装置，其特征在于：所述连接座具有沿所述转动中心线的延伸方向相接的第一部与第二部，所述第一部的外径大于所述第二部的外径，所述第一部朝向所述第二部的一侧为所述凸面，每个所述限位件均能够绕自身轴心线相对旋转地设置在所述第一侧壁上，每个所述限位件的外周面均抵靠在所述第二部的外周面上，所述限位件与所述第二部滚动接触。
  17. 根据权利要求9-16任一权利要求所述的流式电穿孔装置，其特征在于：所述流式电穿孔装置还包括能够绕自身轴心线相对旋转地与所述主机相连的限位件，多个所述限位件间隔设置。
  18. 根据权利要求17所述的流式电穿孔装置，其特征在于：所述主机具有第一侧壁，所述转动座能够绕所述转动中心线相对转动地与所述第一侧壁相连接，所述限位件与所述第一侧壁相连，所述连接座沿所述转动中心线的延伸方向穿过所述第一侧壁。
  19. 根据权利要求17所述的流式电穿孔装置，其特征在于：所述转动座还包括连接座，所述限位件与所述连接座的外周面相抵靠。
  20. 根据权利要求19所述的流式电穿孔装置，其特征在于：所述限位件与所述连接座的外周面滚动接触。
PCT/CN2023/104615 2022-07-14 2023-06-30 一种流式电穿孔装置 WO2024012261A1 (zh)

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