WO2022143546A1 - 一种流式电转染装置 - Google Patents

一种流式电转染装置 Download PDF

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Publication number
WO2022143546A1
WO2022143546A1 PCT/CN2021/141722 CN2021141722W WO2022143546A1 WO 2022143546 A1 WO2022143546 A1 WO 2022143546A1 CN 2021141722 W CN2021141722 W CN 2021141722W WO 2022143546 A1 WO2022143546 A1 WO 2022143546A1
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Prior art keywords
flow
fluid
chamber
electrotransfection
block
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PCT/CN2021/141722
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English (en)
French (fr)
Inventor
戴晓兵
朱士英
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苏州壹达生物科技有限公司
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Application filed by 苏州壹达生物科技有限公司 filed Critical 苏州壹达生物科技有限公司
Priority to US18/270,066 priority Critical patent/US20240067912A1/en
Priority to EP21914282.5A priority patent/EP4273218A1/en
Publication of WO2022143546A1 publication Critical patent/WO2022143546A1/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
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/40—Manifolds; Distribution pieces
    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/18—Flow directing inserts
    • C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
    • 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

Definitions

  • the present invention claims the priority of the Chinese patent application with the application number CN202011587236.1 filed on December 29, 2020.
  • the present invention relates to a flow electrotransfection device, and more particularly, the present invention relates to a device for regulating the flow rate of fluid in the flow electrotransfection device.
  • the method of flow rate at the site to achieve an overall uniform flow rate.
  • the cell membrane is a thin film that surrounds the periphery of the cell and is a permeability barrier for the selective exchange of substances between the cell and the outside world.
  • the cell membrane makes the cell an independent living unit and has a relatively stable internal environment. Some substances in the surrounding environment can pass through the cell membrane, others cannot. Cells can take in nutrients from the surrounding environment through the cell membrane, and excrete metabolites, so that the transport of substances reaches a state of equilibrium. Therefore, the basic function of the cell membrane is to maintain the relative stability of the intracellular microenvironment and selectively exchange substances with the external environment.
  • electrotransfection of cells involves disruption of the lipid bilayer of the cell membrane, resulting in the formation of temporary micropores in the membrane that allow exogenous molecules to enter the cell .
  • a parallel plate electrode or needle electrode array is used to apply an electric field to the cell suspension solution in the chamber.
  • this type of device is called a flow electrotransfection device.
  • the distance between parallel-plate electrodes is usually about 0.2-0.8 cm, that is, the height of the electrotransfection chamber is not large, the length and width (0.5-1 cm) of the electrodes are often relative to each other. Larger, i.e. the length and width of the electrotransfection chamber are also relatively large; the length of the electrotransfection chamber containing the needle electrode array is also large; the properties of the fluid make the fluid flow through the electrotransfection chamber at different sites The fluid flow rate is not uniform, especially in longer chambers.
  • the suspension solution of cells enters and flows through the electrotransfection chamber from the liquid inlet, receives electric shocks from the electrodes in the chamber within a certain time interval, and then flows out from the liquid outlet.
  • the characteristics of the fluid are that the flow rate of the fluid in the electrotransfection chamber is greater near the central region, and the flow rate of the fluid near the wall of the chamber is less.
  • the uneven flow rate of the fluid in the electrotransfection chamber will cause the cells to receive different times of electric shocks, which will affect the electrotransfection effect. If part of the fluid in the electrotransfection chamber is stagnant, the cells in the stagnant area will be subjected to repeated or even sustained electric shocks, causing the cells in the stagnant area to die, thereby affecting the overall cell viability.
  • the present invention needs to solve the technical problem that the fluid flow velocity in the chamber of the existing flow electrotransfection device is not uniform, that is, the fluid velocity in the central area of the chamber is relatively fast, while the fluid velocity near the chamber wall is relatively slow, resulting in a stagnant area, as shown in the figure. 12 shown.
  • the present invention overcomes the above-mentioned deficiencies in the prior art, and provides a flow electrotransfection device, which can make the fluid flow rates of different sites in the electrotransfection chamber tend to be consistent, and the flow rate of fluids passing through different sites in the electrotransfection chamber Cells were shocked by the electrodes for approximately the same number of times, thereby greatly improving cell viability and electrotransfection efficiency.
  • the present invention adopts following technical scheme:
  • a flow electrotransfection device comprising a chamber for fluid to pass through, a liquid inlet channel communicated with the chamber, and a liquid outlet channel communicated with the chamber, the flow electrotransfection device comprising: An electrode assembly for applying an electric field to a fluid in the chamber, the flow electrotransfection device further comprising a fluid guide assembly for equalizing the flow rate of the fluid flowing through the central and edge regions of the chamber .
  • the fluid guide assembly is located in the liquid inlet channel, and the fluid guide assembly includes a plurality of edge distribution blocks and a center distribution block located between the edge distribution blocks, each of the edge distribution blocks. and the central dividing block respectively have an upstream end that is closer to the liquid inlet channel and a downstream end that is farther away from the liquid feeding channel, and the upstream end of the central dividing block and the upstream end of the edge dividing block are between. There is a first gap for fluid to pass between them, and a second gap for fluid to pass between the upstream end of the edge dividing block and the wall defining the liquid inlet channel.
  • the outer contours of the cross-sections of the edge shunt block and the center shunt block consist of arcs.
  • the width of the cross-section of the edge shunt block first increases and then gradually decreases from the upstream end to the downstream end, and the width of the cross-section of the center shunt block starts from its upstream From the end to the downstream end, it increases gradually and then decreases gradually.
  • the width of the cross section of the central shunt block specifically varies according to the following law: the rate of change from the upstream end to the middle portion is greater than the rate of change from the middle portion to the downstream end. That is to say, the width of the portion from the middle to the downstream end (the second half) of the central shunt block changes more gently than the portion from the upstream end to the middle (the first half).
  • the distance between the upstream ends of each of the edge diverting blocks is smaller than the distance between the downstream ends.
  • the distance between the geometric center of each edge distribution block and the geometric center of the central distribution block is equal.
  • liquid inlet channel and the liquid outlet channel are respectively located on the front and rear sides of the chamber, and the upstream end of the central distribution block is located behind the upstream end of the edge distribution block.
  • the upstream end of the central shunt block is located behind the downstream end of the edge shunt block.
  • the flow electrotransfection device further includes an electrode support, the electrode assembly is disposed on the electrode support, and the electrode assembly includes two plate-shaped electrodes, The two electrodes are opposite and spaced apart, the chamber is formed between the electrode support and the two electrodes, and the fluid guide assembly is composed of two edge shunt blocks and one of the center shunt blocks , the edge shunt blocks and the center shunt block are located upstream of the electrode chamber.
  • the liquid inlet channel is formed in a liquid inlet pipe, the liquid inlet pipe is connected to the electrode support, and the edge distribution block and the central distribution block are connected to the inlet.
  • the liquid inlet pipe has a bell mouth portion, the inner wall of the bell mouth portion gradually expands outward along the fluid flow direction, and the edge distribution block and the center distribution block are arranged on the horn. Within the mouth and/or at the region of the chamber adjacent to the flare.
  • the liquid outlet channel is formed in a liquid outlet pipe, the liquid outlet pipe is connected with the electrode support, and the edge distribution block and the central distribution block are connected to the outlet.
  • the fluid guide assembly is located within the inlet channel or at a region of the chamber adjacent to the inlet channel; optionally, the fluid guide assembly is located within the outlet channel or at the region of the chamber adjacent to the outlet channel.
  • the fluid guide assembly includes one or more diverter blocks.
  • the shape of the cross section of the shunt block is a triangle, a trapezoid, a parallelogram, a polygon, a circle, an ellipse, a wavy line or a cone.
  • the fluid guide assembly includes diverter vanes.
  • the fluid guide assembly includes a plurality of the diverter vanes, one end of each of the diverter vanes is connected to each other and the other end extends outward in a radial shape.
  • the electrode assembly comprises an array of parallel plate electrodes or needle electrodes.
  • the present invention adopts the above scheme, has the following advantages compared with the prior art:
  • the flow electrotransfection device of the present invention adopts a flow electrode structure that can realize shunting, which solves the problem of uneven fluid flow velocity at different points in the flow electrotransfection chamber, that is, the flow velocity in the central area of the chamber is faster, and the flow rate near the chamber is faster.
  • the fluid flow rate of the wall is slow and there may be technical problems in the stagnant area, which avoids the phenomenon that the cells in the stagnant area are repeatedly shocked due to long-term accumulation, improves the accuracy of the number of electric shocks, and reduces the damage caused by too many electric shocks. cell damage and even death, thereby improving cell viability and electrotransfection efficiency.
  • Fig. 1 is the exploded structure schematic diagram of the flow electrotransfection device of the embodiment of the present invention.
  • Figure 2 (A) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • FIG. 2(B) is a schematic diagram of the position design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Figure 2 (C) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Figure 2(D) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Figure 2(E) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Fig. 2(F) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Fig. 2(G) is a schematic diagram of the position design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Fig. 2(H) is a schematic diagram of the positional design and cross-sectional shape structure between the diverter blocks in the fluid guide assembly according to the embodiment of the present invention
  • Figure 2 (I) is a schematic diagram of the positional design and cross-sectional shape structure between the shunt blocks in the fluid guide assembly of the embodiment of the present invention
  • Fig. 2(J) is a schematic diagram of the positional design and cross-sectional shape structure between the shunt blocks in the fluid guide assembly according to the embodiment of the present invention
  • FIG. 3 is a cross-sectional view of the fluid guide assembly of the flow electrotransfection device according to the embodiment of the present invention disposed at the liquid inlet;
  • FIG. 4 is a cross-sectional view of a fluid guide assembly of a flow electrotransfection device according to an embodiment of the present invention, which is disposed at a liquid outlet;
  • FIG. 5 is a cross-sectional view of the fluid guide assembly of the flow electrotransfection device according to the embodiment of the present invention, which is disposed at the liquid inlet and the liquid outlet;
  • FIG. 6 is a schematic diagram of a disassembled structure of a flow electrotransfection device according to an embodiment of the present invention.
  • Fig. 7 is the flow electrotransfection device A of Fig. 2(F) and the flow electrotransfection device B without the fluid guiding component, respectively, for CHO-S cells in a buffer containing FITC-Dextran (average molecular weight 500kDa) The experimental results of electrotransfection in ;
  • Fig. 8 shows the experimental results of electrotransfection of plasmid pcDNA3.1 on CHO-S cells using the flow electrotransfection device A of Fig. 2(H) and the flow electrotransfection device B without the fluid guide assembly, respectively. ;
  • FIG. 9 is a schematic diagram of flow velocity distribution in a flow electrotransfection device according to another embodiment of the present invention.
  • FIG. 10 is an exploded view of a flow electrotransfection device according to another embodiment of the present invention, wherein the fluid guide assembly is not shown;
  • Fig. 11 is a partial enlarged view of Fig. 9;
  • FIG. 12 is a schematic diagram of flow velocity distribution in a flow electrotransfection device in the prior art.
  • 1-electrode support 2-first electrode, 3-second electrode, 4-liquid outlet channel, 40-liquid outlet pipe, 5-distribution block, 51-edge distribution block, 511-upstream end, 512- Downstream end, 52-center split block, 521-upstream end, 6-guide opening, 7-fluid guide assembly, 8-liquid inlet channel, 80-liquid inlet pipe, 81-bell mouth, 810-wall, 9-cavity Chamber, 10-Retention Area, 11-Central Spindle, 12-Split Rotary Vane Support Structure, 13-Split Rotary Vane, 100-Fluid.
  • the flow electrotransfection device of this embodiment is shown in FIG. 1 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ; The second electrode 3 is placed in parallel; the electrode support 1 is provided with a liquid inlet channel 8 and a liquid outlet channel 4 respectively, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • FIG. 1 The electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrode 3 is placed in parallel;
  • the electrode support 1 is provided with a liquid inlet channel 8 and a liquid outlet channel 4 respectively, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • FIG. 1 The electrotransfection device consists of an electrode support 1 , a first electrode 2
  • the fluid guide assembly 7 consists of Two or more shunt blocks 5 with a triangular cross-section are arranged and combined, or the shunt blocks 5 with a triangular cross-section and a trapezoidal cross-section are arranged and combined, and a guide opening 6 is formed between the shunt blocks 5.
  • the arrangement of the shunt blocks 5 is as follows: Figure 2(A) and Figure 2(B).
  • the fluid When the fluid flows in from the liquid inlet channel 8, it contacts the fluid guide assembly 7, and a part of the fluid flows to the areas on both sides through the shunt blocks 5 on both sides, and drives the fluid in the retention areas 10 on both sides to flow to the liquid outlet channel 4;
  • the inflowing fluid passes through the guide opening 6 in the middle, so that the fast liquid inflow near the central area in the chamber 9 is slowed down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flows in the chamber 9 as a whole.
  • the fluid flow rate of each site is uniform and smooth, and the fluid electrotransfection is more efficient and effective.
  • the flow electrotransfection device of this embodiment is shown in FIG. 1 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4, and the liquid inlet channel (8 is provided with a fluid guide assembly 7 near the chamber 9, as shown in FIG. 3, the fluid guide assembly 7.
  • the fluid When the fluid flows in from the liquid inlet channel 8), it contacts the fluid guide assembly 7, and a part of the fluid flows to the areas on both sides through the flow distribution blocks 5 on both sides, and drives the fluid in the retention areas 10 on both sides to flow to the liquid outlet channel 4;
  • the fluid flowing directly in passes through the guide opening 6 in the middle, so that the rapid liquid inflow near the central area in the chamber 9 is slowed down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flows in the chamber as a whole.
  • the fluid flow rate at each position in 9 is uniform and smooth, and the fluid electrotransfection is more sufficient and effective.
  • the flow electrotransfection device of this embodiment is shown in FIG. 1 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • FIG. 1 The electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • FIG. 1 The electrotransfection device consists of an electrode support
  • the fluid guide assembly 7 By the arrangement and combination of the shunt blocks 5 whose cross section is a circle and a triangular cross section, or by the arrangement and combination of the shunt blocks 5 whose cross section is a circle and a circular arc transition streamline, the arc-shaped shunt block 5 is in the middle. One or more guide openings 6 are opened, and guide openings 6 are formed between the shunt blocks 5.
  • the arrangement of the shunt blocks is shown in Figures 2(E) and 2(F).
  • the fluid When the fluid flows in from the liquid inlet channel 8, it contacts the fluid guide assembly 7, and a part of the fluid flows to the two side areas through the two-side diverter blocks 5, which drives the fluid in the stagnant areas 10 on both sides to flow to the liquid outlet channel 4, and part of the fluid flows directly from the liquid inlet channel 8.
  • the inflowing fluid passes through the guide opening 6 in the middle, so that the fast liquid inflow near the central area in the chamber 9 is slowed down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flows in the chamber 9 as a whole.
  • the fluid flow rate at each position is uniform and smooth, and the fluid electrotransfection is more efficient and effective.
  • the flow electrotransfection device of this embodiment is shown in FIG. 1 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • the fluid guide assembly 7 Two or more shunt blocks 5 with wavy cross-sections are arranged and combined, and guide openings 6 are formed between the shunt blocks 5.
  • the arrangement of the shunt blocks 5 is shown in Figures 2(G) and 2(H).
  • the fluid When the fluid flows in from the liquid inlet channel 8, it contacts the fluid guide assembly 7, and a part of the fluid flows to the two side areas through the two-side diverter blocks 5, which drives the fluid in the stagnant areas 10 on both sides to flow to the liquid outlet channel 4, and part of the fluid flows directly from the liquid inlet channel 8.
  • the inflowing fluid passes through the guide opening 6 in the middle, so that the fast liquid inflow near the central area in the chamber 9 is slowed down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flows in the chamber 9 as a whole.
  • the fluid flow rate at each position is uniform and smooth, and the fluid electrotransfection is more efficient and effective.
  • the flow electrotransfection device of this embodiment is shown in FIG. 1 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4, and the liquid inlet channel 8 is provided with a fluid guide assembly 7 near the chamber 9.
  • the fluid guide assembly 7 Two or more shunt blocks 5 with irregular polygonal cross-sections are arranged and combined, and guide openings 6 are formed between the shunt blocks 5.
  • the arrangement of the shunt blocks 5 is shown in Figures 2(I) and 2(J).
  • the fluid When the fluid flows in from the liquid inlet channel 8, it contacts the fluid guide assembly 7, and a part of the fluid flows to the two side areas through the two-side diverter blocks 5, which drives the fluid in the stagnant areas 10 on both sides to flow to the liquid outlet channel 4, and part of the fluid flows directly from the liquid inlet channel 8.
  • the inflowing fluid passes through the guide opening 6 in the middle, so that the fast liquid inflow near the central area in the chamber 9 is slowed down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flows in the chamber 9 as a whole.
  • the fluid flow rate at each position is uniform and smooth, and the fluid electrotransfection is more efficient and effective.
  • the position of the fluid guide assembly 7 in the first embodiment to the fifth embodiment can be changed, and the position of the fluid guide assembly 7 is only provided at the liquid outlet channel 4, and the fluid guide assembly 7 is provided at the position 9 of the liquid outlet channel 4 close to the chamber.
  • the fluid guide assembly 7 shown in FIG. 2(F) is arranged at the liquid outlet channel 4, as shown in FIG. 4 .
  • the position of the fluid guide assembly 7 in the first embodiment to the fifth embodiment can be replaced, and both are provided in the liquid inlet channel 8 and the liquid outlet channel 4.
  • the liquid inlet channel 8 is provided with the fluid guide assembly 7 near the chamber 9, and the liquid outlet channel 4
  • a fluid guide assembly 7 is provided near the chamber 9 .
  • the fluid guide assembly 7 shown in FIG. 2(F) is arranged at the liquid inlet channel 8 and the liquid outlet channel 4, as shown in FIG. 5 .
  • the flow electrotransfection device of this embodiment is shown in FIG. 6 .
  • the electrotransfection device consists of an electrode support 1 , a first electrode 2 and a second electrode 3 to form a chamber 9 ; the first electrode 2 and the second electrode 3 form a chamber 9 ;
  • the second electrodes 3 are placed in parallel; the two sides of the electrode support 1 are respectively provided with a liquid inlet channel 8 and a liquid outlet channel 4 , and the liquid inlet channel 8 is provided with a splitter vane support structure 12 near the chamber 9 , and the splitter vane support structure 12 is provided with The central rotating shaft 11 and the splitter vanes 13 .
  • the fluid When the fluid flows in from the liquid inlet channel 8, it contacts the support structure 12 of the splitter rotor. Through the support structure of the splitter rotor 12, the flow of the fluid will drive the splitter rotor 13 to rotate.
  • the fluid flowing out of the flow rotor 13 will flow in the entire chamber 9, driving the fluid in the stagnant areas 10 on both sides to flow to the liquid outlet channel 4, and the fluid flowing directly from the liquid inlet channel 8 will pass through the rotation of the split rotor blade 13 to make the chamber
  • the fast liquid inlet near the central area in 9 slows down, and will not directly rush from the liquid inlet channel 8 to the liquid outlet channel 4, so that the fluid flow rate of the fluid at each point in the chamber 9 is uniform and smooth as a whole, and the fluid is electrotransfected. more effective.
  • the movement of the vertical flow direction is converted into the movement of the helical flow by the splitter vanes 13 , so as to realize the uniformity of the fluid flow rate at each point in the chamber 9 .
  • a control experiment was set up with the flow electrotransfection device A of the embodiment and the flow electrotransfection device B without the fluid guide assembly, and the difference in electrotransfection efficiency was compared. At the same time, a blank control group was set up. The treatment method of the blank control group was the same as that of the electrotransfection group except that no electric shock was performed.
  • CHO-S cells were treated in buffer containing FITC-Dextran (average molecular weight 500kDa) using the flow electrotransfection device A shown in Fig. electrotransfection in solution.
  • Cells in logarithmic growth phase were collected, centrifuged at 300 g for 5 min, and the supernatant was discarded, resuspended and washed with DPBS, and then centrifuged at 300 g for 5 min to discard the supernatant. Resuspend the cells with EBEL electrotransfer buffer to a density of 1 ⁇ 10 8 /mL, then add FITC-Dextran to a final concentration of 0.5mg/mL, and immediately use the devices with A and B for electrotransfection (electroporation) immediately after mixing.
  • FITC-Dextran average molecular weight 500kDa
  • Transfection conditions voltage 220V, pulse width 1ms, interval 1s, times 3). After electrotransfection, the cells were inoculated at a viable cell density of 5 ⁇ 10 6 /mL and cultured in shake flasks. After 2 h, the histogram of cellular FITC signals between different treatment groups was detected by flow cytometry.
  • Fig. 7 show that the cells electrotransfected using the flow electrotransfection device A of Fig. 2(F) in Example 3 of the present invention have a narrower FITC signal peak width, indicating that the number of electric shocks received by the cells is more accurate, Therefore, the transfection efficiency of FITC-Dextran is higher.
  • a control experiment was set up between the flow electrotransfection device A of the embodiment and the flow electrotransfection device B without the fluid guide assembly, and the differences in the cell viability and the GFP positive rate during flow electrotransfection were compared.
  • a blank control group was set up.
  • the treatment method of the blank control group was the same as that of the electrotransfection group except that no electric shock was performed.
  • the CHO-S cells were electrotransfected with the plasmid pcDNA3.1 using the flow electrotransfection device A shown in Figure 2(H) in Example 4 and the flow electrotransfection device B without the fluid guide assembly. Cells in logarithmic growth phase were collected, centrifuged at 300 g for 5 min, and the supernatant was discarded, resuspended and washed with DPBS, and then centrifuged at 300 g for 5 min to discard the supernatant.
  • the cells Resuspend the cells with EBEL electrotransfer buffer to a density of 1 ⁇ 10 8 /mL, then add pcDNA3.1 to a final concentration of 100 ⁇ g/mL, and immediately use the devices with A and B for electrotransfection (electroporation) immediately after mixing.
  • Dyeing conditions voltage 230V, pulse width 600us, interval 1s, times 4).
  • the cells were inoculated at a viable cell density of 4 ⁇ 10 6 /mL, and cultured in shake flasks. After 24 hours, the cells were harvested for 7AAD staining, the cell viability was detected by flow cytometry, and the GFP positive rate of cells in different groups was detected.
  • Fig. 8 show that the cells electrotransfected using the flow electrotransfection device A shown in Fig. 2(H) in Example 4 of the present invention have higher cell viability and GFP positive rate.
  • the flow electrotransfection device of this embodiment is shown in FIGS. 9 to 11 .
  • the flow electrotransfection device has a chamber 9 for fluid 100 to pass through, a liquid inlet channel 8 communicated with the chamber 9 and a liquid outlet channel 4 communicated with the chamber 9.
  • the flow electrotransfection device further includes an electrode support 1 and an electrode assembly for applying an electric field to the fluid 100 in the chamber 9 .
  • the electrode assembly is arranged on the electrode support member 1, and the electrode assembly specifically includes a first electrode 2 and a second electrode 3 in the shape of a plate.
  • the first electrode 2 and the second electrode 3 are opposite to each other and are arranged at intervals. 2 and the chamber 9 formed between the second electrode 3.
  • the liquid inlet channel 8 is formed in a liquid inlet pipe 80 , which is connected with the electrode support 1 ; the liquid outlet channel 4 is formed in a liquid outlet pipe 40 , and the liquid outlet pipe 40 is connected with the electrode support 1 .
  • the liquid inlet pipe 80 and the liquid outlet pipe 40 are respectively connected to opposite sides of the electrode support 1 , such as the left side and the right side as shown in FIG. 9 , or the lower side and the upper side as shown in FIG. 10 .
  • the flow electrotransfection device also includes a fluid guide assembly 7 for making the flow rate of the fluid 100 flowing through the central region and the edge region of the chamber 9 to be uniform.
  • the liquid inlet pipe 80 has a bell mouth portion 81 , the inner wall 810 of the bell mouth portion 81 gradually expands outward along the fluid flow direction, and the fluid guide assembly 7 is disposed on the bell mouth portion 81 .
  • the fluid guide assembly 7 includes two edge distribution blocks 51 and a center distribution block 52 located between the two edge distribution blocks 51 . As shown in FIG.
  • each edge distribution block 51 has an upstream end 511 that is closer to the liquid inlet channel 8 and a downstream end 512 that is farther away from the liquid inlet channel 8
  • the central distribution block 52 has an upstream end 511 that is closer to the liquid inlet channel 8 , respectively.
  • the walls (specifically, the inner wall 810 of the bell mouth portion 81 of the liquid inlet pipe 8 ) respectively have second gaps w1 for fluid to pass through.
  • the edge distribution block 51 and the central distribution block 52 are connected to the inner wall of the liquid inlet pipe 8 so as to be firmly in the liquid inlet channel.
  • the upstream end 521 of the center split block 52 is located behind the upstream end 511 of the edge split block 51 , preferably the downstream end 512 of the edge split block 51 .
  • the orientation words "front” and “rear” are defined according to the flow direction of the fluid as a whole, with the upstream side as “front” and the downstream side as “rear”.
  • the outer contour of the cross-section S1 of the edge shunt block 51 is composed of arcs
  • the outer contour of the cross-section S2 of the center shunt block 52 is composed of arcs.
  • the width of the cross-section S1 of the edge shunt block 51 gradually increases from the upstream end 511 to the downstream end 512 and then gradually decreases
  • the width of the cross-section S2 of the center shunt block 52 gradually increases from the upstream end 521 to the downstream end.
  • the distance between the geometric center of each edge distribution block 51 and the geometric center of the central distribution block 52 is equal.
  • the rate of change of the cross section S2 of the central shunt block 52 from the upstream end 521 to the middle portion is approximately the same as the rate of change from the middle portion to the downstream end, and thus has a flat oval shape.
  • the width of the cross section S2 of the central shunt block 52 varies according to the following law: the rate of change from the upstream end 521 to the middle is greater than the rate of change from the middle to the downstream end. That is to say, the width of the portion from the middle to the downstream end (the second half) of the central shunt block 52 changes more gently than the portion from the upstream end to the middle (the first half), so as to further reduce the width of the central shunt block 52 .
  • the extent of the vortex in the middle region is to be the rate of change from the upstream end 521 to the middle.
  • the process of fluid flowing through the flow electrotransfection device is simulated.
  • the fluid in the liquid inlet channel 8 is first divided into three tributaries by the upstream ends 511 of the two edge diverting blocks 51 , and the middle tributary
  • the flow is further divided by the downstream central diverting block 52, and then the diversion is conducted through the outer peripheral surface of each diverting block to form a distribution as shown in FIG. 9 .
  • the simulation results show that the fluid 100 can flow through the chamber 9 at a relatively uniform flow rate after the bell mouth portion 81 is split and diverted through the three diverting blocks, especially the flow rate of the fluid 100 in the center of the chamber 9 is the same as the chamber 9
  • the flow velocity of the fluid 100 at the edge tends to be uniform, and there is no obvious eddy current, so the effect of making the flow velocity of the fluid uniform is better.
  • the terms “comprising” and “comprising” only imply that the clearly identified steps and elements are included, and these steps and elements do not constitute an exclusive list.
  • the method or apparatus may also include other steps or elements.
  • the term “and/or” includes any combination of one or more of the associated listed items.
  • first”, “second”, etc. are used to describe various information, but the information should not be limited to these terms. These terms are only used to distinguish the same type of information from one another, and do not imply a particular order or level of importance.
  • the expressions “first”, “second” etc. are used completely interchangeably.
  • the first information may also be referred to as the second information
  • the second information may also be referred to as the first information, without departing from the scope of the present disclosure.

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Abstract

一种流式电转染装置，具有用于供流体（100）通过的腔室（9）、与所述腔室（9）连通的进液通道（8）及与所述腔室（9）连通的出液通道（4），所述流式电转染装置包括用于对所述腔室（9）内的流体（100）施加电场的电极组件，所述流式电转染装置还包括用于使流经所述腔室（9）中心区域和边缘区域的流体（100）的流速趋于一致的流体导向组件（7）。解决了流式电转染电极腔室（9）内不同位点的流体（100）流速相对不均匀的技术问题，提高了流体（100）经过电极腔室（9）时受到电击次数的精确性，提高了电转染效率，同时降低了因过多次电击而造成的细胞损伤甚至死亡，从而提高了细胞存活率。

Description

一种流式电转染装置
本发明要求2020年12月29日提交的申请号为CN202011587236.1的中国专利申请的优先权。
技术领域
本发明涉及一种流式电转染装置，更具体而言，本发明涉及一种调控流式电转染装置中流体流速的装置，将流体导向组件用于调控流体在电转染腔室内不同位点的流速以达到整体均匀流速的方法。
背景技术
细胞膜是包围在细胞外周的一层薄膜，是细胞与外界进行选择性物质交换的通透性屏障。细胞膜使细胞成为一个独立的生命单位，并拥有一个相对稳定的内环境。周围环境中的一些物质可以通过细胞膜，其它的物质则不行。细胞可以通过细胞膜从周围环境摄取养料，排出代谢产物，使物质的转运达到平衡状态。所以，细胞膜的基本功能就是维持细胞内微环境的相对稳定并有选择地与外界环境进行物质交换。
研究发现，如果对细胞施加一定强度的电场并持续一段时间，就可以诱导细胞膜上产生一些微孔，使细胞的通透性增强，所谓细胞电穿孔就是指细胞在外加脉冲电场的作用下，细胞膜脂双层上形成瞬时微孔的生物物理过程。当细胞膜发生电穿孔时，其通透性和膜电导会瞬时增大，使亲水分子、核酸、蛋白质、病毒颗粒、药物颗粒等正常情况下不能通过细胞膜的物质得以进入细胞。在短时间内撤除电场后，细胞膜可以自我恢复，重新成为选择性通透屏障。这种利用细胞膜电穿孔的特性将外源物质导入细胞的过程称为细胞电转染。
虽然电转染作用的机理并不完全清楚，但在本文中细胞电转染是公知的，包括细胞膜脂双层的破裂，导致在膜上形成暂时性的微孔，允许外源性分子进入细胞。
根据已有的研究，为了能够实现高通量的细胞电转染过程，在使细胞的悬浮溶液于腔室中流动的同时，采用平行板电极或针电极阵列对腔室内细胞的悬浮溶液施加电场，使其中的细胞受到电击，通常该类装置称为流式电转染装置。
在现有技术公开的装置中，虽然平行板电极之间的距离通常在0.2-0.8厘米左右，即电转染腔室的高度不大，但是电极的长度和宽度(0.5-1厘米)往往相对较大，即电转染腔室的长度和宽度也相对较大；含有针电极阵列的电转染腔室的长度也较大；流体的特性使流体流经电转染腔室时不同位点的流体流速不均匀，尤其在较长的腔室内。
流式电转染装置在工作过程中，细胞的悬浮溶液从进液口进入并流经电转染腔室，在一定时间间隔内受到腔室内电极的电击，再从出液口流出。
流体的特性是电转染腔室内靠近中心区域的流体流速较大，而靠近腔室壁的流体流速较小。
电转染腔室内的流体流速不均匀会造成细胞受到不同次数的电击，影响电转染效果。如果电转染腔室内的部分流体出现了滞留，滞留区域的细胞会受到多次重复、甚至持续的电击，造成滞留区域的细胞死亡，从而影响整体细胞存活率。
而电转染腔室内的流速较快的部分流体，可能受到相对较少次数的电击，这部分流体中的细胞的转染效率较低，降低整体细胞转染效率。因此，用户在实际使用流式电转染装置时，希望能够调整电转染腔室中流体在不同位点的流速，使其尽可能保持一致，以解决上述因流体流式不均匀导致的电转染效率和细胞存活率较低的技术问题。目前尚未有提高流式电转染装置中的流体流速均匀性的现有技术。
本发明需要解决已有的流式电转染装置腔室内流体流速不均匀，即腔室中心区域流体流速较快，而靠近腔室壁的流体流速较慢而产生滞留区域的技术问题，如图12所示。
发明内容
本发明克服了上述现有技术中的不足，提供了一种流式电转染装置，能够使电转染腔室内不同位点的流体流速趋于一致，经过电转染腔室不同位点的细胞受到电极电击次数接近相同，从而大大提高细胞存活率和电转染效率。
为达到上述的目的，本发明采用如下技术方案：
一种流式电转染装置，具有用于供流体通过的腔室、与所述腔室连通的进液通道及与所述腔室连通的出液通道，所述流式电转染装置包括用于对所述腔室内的流体施加电场的电极组件，所述流式电转染装置还包括用于使流经所述腔室中心区域和边缘区域的流体的流速趋 于一致的流体导向组件。
在一实施例中，所述流体导向组件位于所述进液通道内，所述流体导向组件包括多个边缘分流块及位于所述边缘分流块之间的中心分流块，各所述边缘分流块和所述中心分流块分别具有距所述进液通道较近的上游端及距所述进液通道较远的下游端，所述中心分流块的上游端和所述边缘分流块的上游端之间具有供流体通过的第一间隙，所述边缘分流块的上游端与限定所述进液通道的壁之间具有供流体通过的第二间隙。
在一优选的实施例中，两个所述边缘分流块的上游端之间具有第三间隙，所述第三间隙与各所述第二间隙宽度大体相等。
在一优选的实施例中，所述边缘分流块和所述中心分流块的横截面的外轮廓由弧线组成。
在一优选的实施例中，所述边缘分流块的所述横截面的宽度自其上游端至下游端先逐渐增大再逐渐减小，所述中心分流块的所述截面的宽度自其上游端至下游端先逐渐增大再逐渐减小。
在一优选的实施例中，所述中心分流块的所述横截面的宽度具体按照如下规律变化：自上游端至中部的变化率大于自中部至下游端的变化率。也就是说，所述中心分流块的中部至下游端的部分(其后半段)相比其上游端至中部的部分(其前半段)的宽度变化较为平缓。
在一优选的实施例中，各所述边缘分流块的上游端之间的距离小于下游端之间的距离。
在一优选的实施例中，各所述边缘分流块的几何中心与所述中心分流块的几何中心的距离相等。
在一优选的实施例中，所述进液通道及所述出液通道分别位于所述腔室的前后两侧，所述中心分流块的上游端位于所述边缘分流块的上游端的后侧。
在一优选的实施例中，所述中心分流块的上游端位于所述边缘分流块的下游端的后侧。
在一具体且优选的实施例中，所述流式电转染装置还包括电极支撑件，所述电极组件设置于所述电极支撑件上，所述电极组件包括呈板状的两个电极，两个所述电极相对且间隔设置，所述电极支撑件和两个所述电极之间形成所述腔室，所述流体导向组件由两个所述边缘分流块和一个所述中心分流块组成，所述各边缘分流块与所述中心分流块位于所述电极腔室 的上游。
在一更优选的实施例中，所述进液通道形成于一进液管内，所述进液管和所述电极支撑件连接，所述边缘分流块和所述中心分流块连接于所述进液管的内壁。
在一更优选的实施例中，所述进液管具有喇叭口部，所述喇叭口部的内壁沿流体流动方向逐渐外扩，所述边缘分流块和所述中心分流块设置于所述喇叭口部内和/或所述腔室的邻近所述喇叭口部的区域处。
在一更优选的实施例中，所述出液通道形成于一出液管内，所述出液管和所述电极支撑件连接，所述边缘分流块和所述中心分流块连接于所述出液管的内壁。
在一实施例中，所述流体导向组件位于所述进液通道内或所述腔室的邻近所述进液通道的区域处；可选地，所述流体导向组件位于所述出液通道内或所述腔室的邻近所述出液通道的区域处。
在一实施例中，所述流体导向组件包括一或多个分流块。
在一优选的实施例中，所述分流块的横截面的形状为三角形、梯形、平行四边形、多边形、圆形、椭圆形、波浪线形或锥形。
在一实施例中，所述流体导向组件包括分流叶片。
在一优选的实施例中，所述流体导向组件包括多个所述分流叶片，各所述分流叶片的一端相互连接而另一端向外延伸呈辐射状。
在一实施例中，所述电极组件包括一组平行板电极或针电极阵列。
本发明采用以上方案，相比现有技术具有如下优点：
本发明的流式电转染装置采用可实现分流的流式电极结构，解决了流式电转染腔室内的不同位点流体流速不均匀，即腔室中心区域流速较快，而靠近腔室壁的流体流速慢并可能出现滞留区域的技术问题，避免了滞留区域的细胞因长时间累积而受到多次重复电击的现象，提高了电击次数的精确性，降低了因过多次电击而造成的细胞损伤甚至死亡，从而提高了细胞存活率和电转染效率。
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地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例的流式电转染装置的分解结构示意图；
图2(A)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(B)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(C)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(D)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(E)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(F)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(G)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(H)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(I)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图2(J)为本发明实施例的流体导向组件中分流块之间的位置设计和横截面形状结构示意图；
图3为本发明实施例的流式电转染装置流体导向组件设置于进液口的剖视图；
图4为本发明实施例的流式电转染装置流体导向组件设置于出液口的剖视图；
图5为本发明实施例的流式电转染装置流体导向组件设置于进液口和出液口的剖视图；
图6为本发明实施例的流式电转染装置的分解结构示意图；
图7为利用图2(F)的流式电转染装置A和未使用流体导向组件的流式电转染装置B分别对CHO-S细胞在含有FITC-Dextran(平均分子量500kDa)的缓冲液中进行电转染的实验结果；
图8为利用图2(H)的流式电转染装置A和未使用流体导向组件的流式电转染装置B分别对CHO-S细胞进行了质粒pcDNA3.1的电转染的实验结果；
图9为根据本发明另一实施例的流式电转染装置中的流速分布示意图；
图10为根据本发明另一实施例的流式电转染装置的分解图，其中未示出流体导向组件；
图11为图9的局部放大图；
图12为一种现有技术中的流式电转染装置中的流速分布示意图。
其中，1-电极支撑件，2-第一电极，3-第二电极，4-出液通道，40-出液管，5-分流块，51-边缘分流块，511-上游端，512-下游端，52-中心分流块，521-上游端，6-导向开口，7-流体导向组件，8-进液通道，80-进液管，81-喇叭口部，810-壁，9-腔室，10-滞留区域，11-中心转轴，12-分流转叶支撑结构，13-分流转叶，100-流体。
具体实施方式
下面将对发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例一
本实施例所述流式电转染装置如图1所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1上下分别设 置进液通道8和出液通道4，进液通道8靠近腔室9处设置流体导向组件7，如图3所示，流体导向组件7由两个或多个横截面为三角形的分流块5排列组合，或者由横截面为三角形和横截面为梯形的分流块5排列组合，分流块5之间形成导向开口6，分流块5排列方式如图2(A)和图2(B)所示。
流体从进液通道8流入时，接触到流体导向组件7，一部分流体通过两侧分流块5流向两侧区域，带动两侧滞留区域10的流体流向出液通道4；一部分从进液通道8径直流入的流体通过中部的导向开口6，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位点的流体流速均匀顺畅，流体电转染更充分有效。
实施例二
本实施例所述流式电转染装置如图1所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1两侧分别设置进液通道8和出液通道4，进液通道(8靠近腔室9处设置流体导向组件7，如图3所示，流体导向组件7由两个或多个横截面为四边形的分流块5排列组合，或者由横截面为四边形和横截面为三角形的分流块5排列组合，分流块5之间形成导向开口6，分流块5排列方式如图2(C)和图2(D)所示。
流体从进液通道8)流入时，接触到流体导向组件7，一部分流体通过两侧分流块5流向两侧区域，带动两侧滞留区域10的流体流向出液通道4；一部分从进液通道8径直流入的流体通过中部的导向开口6，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位置点的流体流速均匀顺畅，流体电转染更充分有效。
实施例三
本实施例所述流式电转染装置如图1所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1两侧分别设置进液通道8和出液通道4，进液通道8靠近腔室9处设置流体导向组件7，如图3所示，流体导向组件7由横截面为圆形和横截面为三角形的分流块5排列组合，或者由横截面为圆形 和横截面为圆弧过渡流线形的分流块5排列组合，圆弧形的分流块5中间开一个或多个导向开口6，且分流块5之间形成导向开口6，分流块排列方式如图2(E)和2(F)所示。
流体从进液通道8流入时，接触到流体导向组件7，一部分流体通过两侧分流块5流向两侧区域，带动两侧滞留区域10的流体流向出液通道4，一部分从进液通道8径直流入的流体通过中部的导向开口6，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位置点的流体流速均匀顺畅，流体电转染更充分有效。
实施例四
本实施例所述流式电转染装置如图1所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1两侧分别设置进液通道8和出液通道4，进液通道8靠近腔室9处设置流体导向组件7，如图3所示，流体导向组件7由两个或多个横截面为波浪线形的分流块5排列组合，分流块5之间形成导向开口6，分流块5排列方式如图2(G)和图2(H)所示。
流体从进液通道8流入时，接触到流体导向组件7，一部分流体通过两侧分流块5流向两侧区域，带动两侧滞留区域10的流体流向出液通道4，一部分从进液通道8径直流入的流体通过中部的导向开口6，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位置点的流体流速均匀顺畅，流体电转染更充分有效。
实施例五
本实施例所述流式电转染装置如图1所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1两侧分别设置进液通道8和出液通道4，进液通道8靠近腔室9处设置流体导向组件7，如图3所示，流体导向组件7由两个或多个横截面为不规则多边形的分流块5排列组合，分流块5之间形成导向开口6，分流块5排列方式如图2(I)和图2(J)所示。
流体从进液通道8流入时，接触到流体导向组件7，一部分流体通过两侧分流块5流向两侧区域，带动两侧滞留区域10的流体流向出液通道4，一部分从进液通道8径直流入的流体 通过中部的导向开口6，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位置点的流体流速均匀顺畅，流体电转染更充分有效。
可以更换实施例一至实施例五中流体导向组件7的位置，只在出液通道4处设置，出液通道4靠近腔室处9设置流体导向组件7。例如将图2(F)所示的流体导向组件7设置于出液通道4处，如图4所示。
可以更换实施例一至实施例五中流体导向组件7的位置，在进液通道8和出液通道4两处都设置，进液通道8靠近腔室9处设置流体导向组件7，出液通道4靠近腔室处9设置流体导向组件7。例如将图2(F)所示的流体导向组件7设置于进液通道8和出液通道4处，如图5所示。
实施例六
本实施例所述流式电转染装置如图6所示，所述电转染装置由电极支撑件1、第一电极2和第二电极3组成腔室9；所述第一电极2和第二电极3平行放置；电极支撑件1两侧分别设置进液通道8和出液通道4，进液通道8靠近腔室9处设置分流转叶支撑结构12，分流转叶支撑结构12上设置中心转轴11和分流转叶13。
流体从进液通道8流入时，接触到分流转叶支撑结构12，通过分流转叶支撑结构12，流体的流动会带动分流转叶13转动，分流转叶13绕着中心转轴11旋转，从分流转叶13流出去的流体会在整个腔室9内流动，带动两侧滞留区域10的流体流向出液通道4，从进液通道8径直流入的流体通过分流转叶13的转动，使腔室9内靠近中心区域的快速进液缓慢下来，不会从进液通道8直接冲向出液通道4，从而在整体上使流体在腔室9内各位点的流体流速均匀顺畅，流体电转染更充分有效。
通过分流转叶13将垂直流向的运动转换成螺旋流动的运动，实现腔室9内各位点的流体流速的均匀性。
实施例七
将实施例的流式电转染装置A和未使用流体导向组件的流式电转染装置B设置对照实验，比较电转染效率的差别。同时设置空白对照组，空白对照组除不进行电击外，处理方式与电 转染组实验相同。
利用实施例三中图2(F)的流式电转染装置A和未使用流体导向组件的流式电转染装置B分别对CHO-S细胞在含有FITC-Dextran(平均分子量500kDa)的缓冲液中进行电转染。收集对数生长期的细胞，以300g离心5min后弃除上清，用DPBS重悬清洗，再以300g离心5min弃除上清。用EBEL电转缓冲液重悬细胞至密度为1×10 8/mL，再加入FITC-Dextran至终浓度为0.5mg/mL，混匀后立即分别用带A和B的装置进行电转染(电转染条件：电压220V，脉宽1ms，间隔1s，次数3次)。电转染结束后，以活细胞密度5×10 6/mL进行接种，摇瓶培养。2h后，用流式细胞术检测不同处理组之间细胞FITC信号的直方图。
由图7结果显示，使用本发明施例三中图2(F)的流式电转染装置A进行电转染的细胞，FITC信号峰宽更窄，表明细胞受到的电击次数更为准确，因此对FITC-Dextran的转染效率更高。
实施例八
将实施例流式电转染装置A和未使用流体导向组件的流式电转染装置B设置对照实验，比较流式电转染时细胞的存活率和GFP阳性率的差别。同时设置空白对照组，空白对照组除不进行电击外，处理方式与电转染组实验相同。
利用实施例四中图2(H)的流式电转染装置A和未使用流体导向组件的流式电转染装置B分别对CHO-S细胞进行了质粒pcDNA3.1的电转染。收集对数生长期的细胞，以300g离心5min后弃除上清，用DPBS重悬清洗，再以300g离心5min弃除上清。用EBEL电转缓冲液重悬细胞至密度为1×10 8/mL，再加入pcDNA3.1至终浓度为100μg/mL，混匀后立即分别用带A和B的装置进行电转染(电转染条件：电压230V，脉宽600us，间隔1s，次数4次)。电转染结束后，以活细胞密度4×10 6/mL进行接种，摇瓶培养。24h后，收取细胞进行7AAD染色，用流式细胞术检测细胞的存活率，并检测不同组细胞的GFP阳性率。
由图8结果显示，使用本发明实施例四中图2(H)的流式电转染装置A进行电转染的细胞，细胞存活率和GFP阳性率更高。
实施例九
本实施例流式电转染装置如图9至11所示。该流式电转染装置具有用于供流体100通过 的腔室9、与腔室9连通的进液通道8及与腔室9连通的出液通道4。流式电转染装置还包括电极支撑件1及用于对腔室9内的流体100施加电场的电极组件。电极组件设置于电极支撑件1上，电极组件具体包括呈板状的第一电极2和第二电极3，第一电极2和第二电极3相对且间隔设置，电极支撑件1、第一电极2和第二电极3之间形成的腔室9。进液通道8形成于一进液管80内，进液管80和电极支撑件1连接；出液通道4形成于一出液管40内，出液管40和电极支撑件1连接。进液管80和出液管40分别连接于电极支撑件1的相对两侧部，如图9所示的左侧部和右侧部，或图10所示的下侧部和上侧部。
该流式电转染装置还包括用于使流经腔室9中心区域和边缘区域的流体100的流速趋于一致的流体导向组件7。进液管80具有喇叭口部81，喇叭口部81的内壁810沿流体流动方向逐渐外扩，流体导向组件7设置于喇叭口部81。流体导向组件7包括两个边缘分流块51及位于两个边缘分流块51之间的中心分流块52，两个边缘分流块51及一个中心分流块52均位于喇叭口部81内。如图11所示，各边缘分流块51分别具有距进液通道8较近的上游端511及距进液通道8较远的下游端512，中心分流块52分别具有距进液通道8较近的上游端521及距进液通道8较远的下游端。中心分流块52的上游端521和两侧的边缘分流块51的上游端511之间分别具有供流体通过的第一间隙，两侧的边缘分流块51的上游端511与限定进液通道8的壁(具体为进液管8的喇叭口部81的内壁810)之间分别具有供流体通过的第二间隙w1。边缘分流块51和中心分流块52连接于进液管8的内壁，以稳固地处于进液通道内。
如图11所示，本实施例中，中心分流块52的上游端521位于边缘分流块51的上游端511的后侧，优选地位于边缘分流块51的下游端512的后侧。本文中，方位词“前”“后”依据流体整体的流向定义，以位于上游一侧为“前”，以位于下游一侧为“后”。两个边缘分流块51的上游端511之间具有供流体通过的第三间隙w2，第三间隙w2与两侧的第二间隙w1的宽度大体相等。边缘分流块51的横截面S1的外轮廓由弧线组成，中心分流块52的横截面S2的外轮廓由弧线组成。其中，边缘分流块51的横截面S1的宽度自上游端511至其下游端512先逐渐增大再逐渐减小，中心分流块52的横截面S2的宽度自其上游端521至下游端先逐渐增大再逐渐减小；且各边缘分流块51的几何中心与中心分流块52的几何中心的距离相等。本实施例中，中心分流块52的横截面S2自上游端521至中部的变化率与自中部至下游端的变化率大致相同而呈扁平的椭圆形。在一些实施例中，中心分流块52的横截面S2的宽度具体按照如下规律变化：自上游端521至中部的变化率大于自中部至下游端的变化率。也就是 说，中心分流块52的中部至下游端的部分(其后半段)相比其上游端至中部的部分(其前半段)的宽度变化较为平缓，以进一步地降低中心分流块52后的中间区域涡流的范围。
如图9所示，对流体流经流式电转染装置的过程进行了模拟，进液通道8内的流体先被两个边缘分流块51的上游端511分为三个支流，中间的支流进一步被下游的中心分流块52继续分流，然后通过各个分流块的外周面进行导流，形成如图9所示的分布。模拟结果显示，流体100在喇叭口部81经三个分流块进行分流和导流后，能够以相对均匀的流速流过腔室9，特别是腔室9中心的流体100的流速与腔室9边缘(邻近限定腔室9的壁的区域)的流体100的流速趋于一致，且无明显涡流，使流体流速均匀的效果较优。
如本说明书和权利要求书中所示，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法或者设备也可能包含其他的步骤或元素。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的组合。进一步可以理解的是，术语“第一”、“第二”等用于描述各种信息，但这些信息不应限于这些术语。这些术语仅用来将同一类型的信息彼此区分开，并不表示特定的顺序或者重要程度。实际上，“第一”、“第二”等表述完全可以互换使用。例如，在不脱离本公开范围的情况下，第一信息也可以被称为第二信息，类似地，第二信息也可以被称为第一信息。
上述实施例只为说明本发明的技术构思及特点，是一种优选的实施例，其目的在于熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限定本发明的保护范围。

Claims (20)

  1. 一种流式电转染装置，具有用于供流体通过的腔室、与所述腔室连通的进液通道及与所述腔室连通的出液通道，所述流式电转染装置包括用于对所述腔室内的流体施加电场的电极组件，其特征在于，所述流式电转染装置还包括用于使流经所述腔室中心区域和边缘区域的流体的流速趋于一致的流体导向组件。
  2. 根据权利要求1所述的流式电转染装置，其特征在于，所述流体导向组件位于所述进液通道内，所述流体导向组件包括多个边缘分流块及位于所述边缘分流块之间的中心分流块，各所述边缘分流块和所述中心分流块分别具有距所述进液通道较近的上游端及距所述进液通道较远的下游端，所述中心分流块的上游端和所述边缘分流块的上游端之间具有供流体通过的第一间隙，所述边缘分流块的上游端与限定所述进液通道的壁之间具有供流体通过的第二间隙。
  3. 根据权利要求2所述的流式电转染装置，其特征在于，两个所述边缘分流块的上游端之间具有第三间隙，所述第三间隙与各所述第二间隙宽度大体相等。
  4. 根据权利要求2所述的流式电转染装置，其特征在于，所述边缘分流块和所述中心分流块的横截面的外轮廓由弧线组成。
  5. 根据权利要求4所述的流式电转染装置，其特征在于，所述边缘分流块的所述横截面的宽度自其上游端至下游端先逐渐增大再逐渐减小，所述中心分流块的所述截面的宽度自其上游端至下游端先逐渐增大再逐渐减小。
  6. 根据权利要求5所述的流式电转染装置，其特征在于，所述中心分流块的所述横截面的宽度具体按照如下规律变化：自上游端至中部的变化率大于自中部至下游端的变化率。
  7. 根据权利要求5所述的流式电转染装置，其特征在于，各所述边缘分流块的上游端之间的距离小于其下游端之间的距离。
  8. 根据权利要求2所述的流式电转染装置，其特征在于，各所述边缘分流块的几何中心与所述中心分流块的几何中心的距离相等。
  9. 根据权利要求2所述的流式电转染装置，其特征在于，所述进液通道及所述出液通道分别位于所述腔室的前后两侧，所述中心分流块的上游端位于所述边缘分流块的上游端的后侧。
  10. 根据权利要求9所述的流式电转染装置，其特征在于，所述中心分流块的上游端位于所述边缘分流块的下游端的后侧。
  11. 根据权利要求2至10任一项所述的流式电转染装置，其特征在于，所述流式电转染装置 还包括电极支撑件，所述电极组件设置于所述电极支撑件上，所述电极组件包括呈板状的两个电极，两个所述电极相对且间隔设置，所述电极支撑件和两个所述电极之间形成所述的腔室，所述流体导向组件由两个所述边缘分流块和一个所述中心分流块组成，所述边缘分流块与所述中心分流块位于所述电极的上游。
  12. 根据权利要求11所述的流式电转染装置，其特征在于，所述进液通道形成于一进液管内，所述进液管和所述电极支撑件连接，所述边缘分流块和所述中心分流块连接于所述进液管的内壁。
  13. 根据权利要求12所述的流式电转染装置，其特征在于，所述进液管具有喇叭口部，所述喇叭口部的内壁沿流体流动方向逐渐外扩，所述边缘分流块和所述中心分流块设置于所述喇叭口部内和/或所述腔室的邻近所述喇叭口部的区域。
  14. 根据权利要求12所述的流式电转染装置，其特征在于，所述出液通道形成于一出液管内，所述出液管和所述电极支撑件连接，所述边缘分流块和所述中心分流块连接于所述出液管的内壁。
  15. 根据权利要求1所述的流式电转染装置，其特征在于，所述流体导向组件位于所述进液通道内或所述腔室的邻近所述进液通道的区域处；和/或，所述流体导向组件位于所述出液通道内或所述腔室的邻近所述出液通道的区域处。
  16. 根据权利要求1所述的流式电转染装置，其特征在于，所述流体导向组件包括一个或多个分流块。
  17. 根据权利要求16所述的流式电转染装置，其特征在于，所述分流块的横截面的形状为三角形、梯形、平行四边形、多边形、圆形、椭圆形、波浪线形或锥形。
  18. 根据权利要求1所述的流式电转染装置，其特征在于，所述流体导向组件包括分流叶片。
  19. 根据权利要求18所述的流式电转染装置，其特征在于，所述流体导向组件包括多个所述分流叶片，各所述分流叶片的一端相互连接而另一端向外延伸呈辐射状。
  20. 根据权利要求1所述的流式电转染装置，其特征在于，所述电极组件包括针电极阵列或一组平行板电极。
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