WO2020093374A1 - 细胞电融合培养芯片和装置、细胞电融合的方法 - Google Patents

细胞电融合培养芯片和装置、细胞电融合的方法 Download PDF

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WO2020093374A1
WO2020093374A1 PCT/CN2018/114845 CN2018114845W WO2020093374A1 WO 2020093374 A1 WO2020093374 A1 WO 2020093374A1 CN 2018114845 W CN2018114845 W CN 2018114845W WO 2020093374 A1 WO2020093374 A1 WO 2020093374A1
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culture
cell
electrode
interdigitated
layer
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French (fr)
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王文会
何炜华
黄亮
丁为
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Tsinghua University
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Tsinghua University
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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
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

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  • the present application relates to the field of cell fusion technology. Specifically, the present application relates to a cell electrofusion culture chip, a cell electrofusion culture device, and a cell electrofusion method.
  • polyploid cancer cells are larger than most other cancer cells, are resistant to chemotherapy and radiotherapy, and are associated with disease recurrence. These giant cells are harder than other cancer cells and can move farther, which may help explain why they are associated with more serious diseases.
  • Polyploid cancer cells are usually derived from the abnormal division of cancer cells, and can also be artificially prepared by cell fusion.
  • Cell fusion refers to the fusion of two cells or protoplasts to form a cell under artificial induction or spontaneous conditions. It has important significance in the study of genetic material.
  • Cell electrofusion technology is a method of controlling the fusion of two or more cells into a single-nuclear or multi-nuclear hybrid cell by external electric field. Compared with the induced fusion technology of biology, chemistry and physics, cell electrofusion technology has the advantages of higher efficiency, simple operation, and no toxicity to cells. It has gradually become an important method in the field of bioengineering research.
  • the microfluidic chip has the characteristics of high throughput, very few samples and reagents, so it is widely used in analysis and detection in the fields of biology, chemistry, and medicine.
  • Cell fusion devices based on microfluidic technology can consume a small amount of reagents and achieve stable fusion of a large number of cells in a short time.
  • Dielectrophoresis is the phenomenon of dielectric forces in a non-uniform electric field, and is often used to manipulate tiny particles in fluids. The presence of dielectrophoretic force does not require the object itself to be charged. All particles have a dielectrophoresis phenomenon in an electric field environment. However, the magnitude of the force largely depends on the electrical properties of the substance and particles, the shape and size of the particles, and the rate of change of the field strength. Therefore, an electric field of a specific frequency can selectively control specific particles. With the aid of dielectrophoretic force, we can more easily manipulate cells.
  • the cell electrofusion culture chip designed by the present application can use the principle of dielectrophoresis to efficiently capture and pair cells in an array, use electric pulses to fuse cells that have already been paired, and use the double-well structure to realize the culture analysis of the fused cells.
  • the present application can play an important role in cell genetic research based on cell fusion.
  • the present application proposes a cell electrofusion culture chip.
  • the cell electrofusion culture chip includes: a substrate; at least one first interdigitated electrode and at least one second interdigitated electrode, the at least one first interdigitated electrode and at least one second interdigitated finger Electrodes are provided on the upper surface of the substrate, and each of the first interdigitated electrodes has at least one first protruding electrode, and each of the second interdigitated electrodes has at least one second protruding electrode; the capture layer, The capture layer covers the at least one first interdigitated electrode and at least one second interdigitated electrode, and has at least one capture hole that penetrates the capture layer; the culture layer, the culture layer covers the A capture layer, a cavity is defined between a lower surface of the culture layer and an upper surface of the capture layer, and the culture layer is further provided with two entrances and exits and at least one culture hole, the entrances and exits penetrate the The culture layer is used for the in and out of the cell suspension, the culture hole does not penetrate the culture layer and is arranged directly opposite to the capture hole; wherein,
  • the cell electrofusion culture chip of the embodiment of the present application can use the principle of dielectrophoresis to efficiently capture cells and achieve precise pairing between the first interdigitated electrode and the second interdigitated electrode. Electrofusion provides ideal conditions. It can also use electrical pulses to induce electroporation and change the permeability of cell membranes to achieve cell fusion. Moreover, it can use the principle of negative dielectrophoresis to keep cells away from electrodes during fusion, thereby preventing electrodes from damaging cells. And, the double-well structure of the capture hole and the culture hole can be used to transfer the fused cells to the culture hole under the influence of gravity, and then continue the subsequent culture to analyze the effect of cell fusion.
  • cell electrofusion culture chip according to the above embodiments of the present application may also have the following additional technical features:
  • the material forming the substrate is inorganic glass or organic glass, and the material forming the first and second interdigital electrodes is metal or a conductive polymer, forming the capture layer
  • the material of the photoresist is photoresist, and the material forming the culture layer includes polydimethylsiloxane.
  • the thickness of the first interdigitated electrode and the second interdigitated electrode is 50-150 nanometers
  • the length of the square trapping holes is 10-100 micrometers and the width is 5-50 micrometers
  • the distance between the first protruding electrode and the second protruding electrode in one of the capture holes is 20-30 microns.
  • the cell electrofusion culture chip includes a plurality of first interdigitated electrodes and a plurality of second interdigitated electrodes arranged in parallel with each other, and each of the first interdigitated electrodes Having a plurality of the first protruding electrodes, each of the second interdigitated electrodes has a plurality of the second protruding electrodes;
  • the capture layer has a plurality of the capture holes, and the culture layer has a plurality of The culture holes, and the plurality of capture holes and the plurality of culture holes are all arranged in an array.
  • the number of the capture holes is 50-1000.
  • the capture layer and the culture layer are detachable.
  • the present application proposes a cell electrofusion culture device.
  • the cell electrofusion culture device includes: the above cell electrofusion culture chip; a signal generator, the signal generator and the at least one first interdigitated electrode and at least one second prong respectively
  • the finger electrode is electrically connected and used to send different electrical signals to the cell electrofusion culture chip; a microfluidic pump, which is connected to the two inlets and outlets respectively, is used to control the flow of the cell suspension.
  • the cell electrofusion culture device of the embodiment of the present application can realize the functions of cell capture, cell pairing and cell fusion of the cell electrofusion culture device through different electrical signals of the signal generator, and is controlled by the microfluidic pump Flow of cell suspension.
  • the features and advantages described above for the cell electrofusion culture chip are still applicable to the cell electrofusion culture device, which will not be repeated here.
  • the present application proposes a method of cell electrical fusion.
  • the method uses the above-mentioned cell electrofusion culture device, and the method includes: making the cell suspension enter the cavity from the entrance and exit of the cell electrofusion culture chip through a microfluidic pump; A sinusoidal electrical signal is applied to the first and second interdigitated electrodes, and the cells in the cell suspension enter the capture hole and are adsorbed to the first or second protruding electrode; by adjusting The signal frequency of the signal generator, so that the cells adsorbed by the first protruding electrode and the cells adsorbed by the second protruding electrode are contacted and paired; the first The interdigitated electrode and the second interdigitated electrode apply an electrical pulse signal to cause electroporation and fusion of the contact portion of the cell.
  • the cell electrofusion method of the embodiment of the present application can capture and pair cells accurately and efficiently using the principle of positive dielectrophoresis, improve the stability and accuracy of cell pairing, and use the principle of negative dielectrophoresis to make The cells are kept away from the electrodes during fusion to prevent the electrical pulse signal during fusion from causing the electrodes to cause damage to the cells.
  • the features and advantages described above for the cell electrofusion culture chip and the cell electrofusion culture device are still applicable to the cell electrofusion method, which will not be repeated here.
  • the method further includes: turning the cell electrofusion culture chip 180 ° so that the fused cells fall into the culture well;
  • the microfluidic pump allows the culture medium to enter the culture hole of the cell electrofusion culture chip to culture the fused cells.
  • the number of the electric pulse signals is 5, the pulse width is 50 microseconds, and the frequency is 1 Hz.
  • FIG. 1 is a schematic diagram of an oblique view of a cell electrofusion culture chip according to an embodiment of the present application
  • FIG. 2 is a schematic structural plan view of a cell electrofusion culture chip according to an embodiment of the present application
  • FIG. 3 is a schematic diagram of a cross-sectional structure of a cell electrofusion culture chip according to an embodiment of the present application
  • FIG. 4 is an example diagram and enlarged view of a cell electrofusion culture chip according to another embodiment of the present application:
  • A A photo of the cell electrofusion culture chip actually produced
  • FIG. 5 is a schematic flowchart of a method of cell electrical fusion according to an embodiment of the present application.
  • FIG. 6 is a schematic flowchart of a method of cell electrical fusion according to another embodiment of the present application.
  • FIG. 10 is a simulation diagram of instantaneously capturing the electric field distribution in a hole by applying an electric pulse signal according to an embodiment of the present application
  • FIG. 11 is a photograph of the process of electrical fusion of Hella cells according to an embodiment of the present application
  • FIG. 12 is a photograph of fused cells cultured in culture wells according to an embodiment of the present application.
  • the present application proposes a cell electrofusion culture chip.
  • the cell electrofusion culture chip 100 includes a substrate 110, at least one first interdigitated electrode 120, at least one second interdigitated electrode 130, a capture layer 140, and a culture layer 150; wherein At least one first interdigitated electrode 120 and at least one second interdigitated electrode 130 are provided on the upper surface of the substrate 110, and each first interdigitated electrode 120 has at least one first protruding electrode 121, each The two interdigitated electrode 130 has at least one second protruding electrode 131; the capture layer 140 covers at least one first interdigitated electrode 120 and at least one second interdigitated electrode 130, the capture layer 140 has at least one trap hole 141, and the trap hole 141 penetrates the capture layer 140; the culture layer 150 covers the capture layer 140, a cavity 160 is defined between the lower surface of the culture layer 150 and the upper surface of the capture layer 140, and the culture layer 150 is further provided with two inlets 151 and at least one The culture hole 152, and the inlet and outlet
  • the material for forming the substrate 110 may be inorganic glass or organic glass, the material for forming the first and second interdigital electrodes 120 and 130 may be a metal or conductive polymer, and the material for forming the capture layer 140 It may be photoresist, and the material forming the culture layer 150 includes polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the substrate 110 of transparent material is convenient for observation, and the electrode of the conductive material is easier to polarize the cell into a dipole. Selecting a transparent photoresist material can lithographically capture holes 141 in the replenishment layer 140.
  • the culture layer 150 may be formed by reverse molding with polydimethylsiloxane material.
  • the thickness of the first and second interdigitated electrodes 120 and 130 may be 50 to 150 nanometers, and the length of the square trapping hole 141 may be 10 to 100 micrometers and the width may be 5 to 50 micrometers
  • the distance between the first protruding electrode 121 and the second protruding electrode 131 in one trapping hole 141 may be 20-30 microns. In this way, between the first protruding electrode 121 and the second protruding electrode 131 in the exposed portion of the cavity 160 formed with the above dimensions, a dielectrophoretic effect can be formed to directionally migrate cells.
  • the first and second interdigitated electrodes 120 and 130 can be formed by sputtering and photolithography to form 20-nanometer-thick platinum and 80-nanometer-thick gold. Not easily damaged.
  • the cell electrofusion culture chip 100 may include a plurality of first interdigitated electrodes 120 and a plurality of second interdigitated electrodes 130 arranged in parallel with each other, and each first interdigitated electrode 120 may have multiple Each first protruding electrode 121, each second interdigitated electrode 130 may have a plurality of second protruding electrodes 131; and the capture layer 140 may have a plurality of capture holes 141, and the culture layer 150 may have a plurality of culture holes 152, Moreover, the plurality of capture holes 141 and the plurality of culture holes 152 are all arranged in an array.
  • each culture hole in the culture hole array can be arranged directly opposite to each capture hole in the capture hole array, and a portion of the first protruding electrode and the second protruding electrode are exposed in each capture hole, and refer to Figure 4.
  • the capture holes and culture holes of this large-scale array structure design correspond to the interdigitated electrode array, so that a large number of cells can be captured and electrically fused at the same time.
  • the number of capture holes may be 50-1000, so that thousands of fusion cells can be obtained through one electrofusion process.
  • the capture layer 140 and the culture layer 150 are detachable, specifically, for example, assembled by reversible bonding, etc. In this way, the bottom surface of the culture layer 150 and the capture layer are captured before electrofusion The upper surface of the layer 140 is bonded. After the test batch of cells is captured, the capture layer 140 and the culture layer 150 are disassembled, and after necessary cleaning and disinfection treatments, the synthetic layer is bonded to capture the next batch of cells.
  • the width dimension of the first interdigitated electrode 120 and the second interdigitated electrode 130 can be designed according to the cell size, and the length is designed according to the required flux size; the height dimension of the cavity 160, The matching design is performed according to the cell size; the length, width, and depth of the capture hole 141 are all designed according to the cell size.
  • the present application proposes a cell electrofusion culture chip
  • the first interdigitated electrode and the second interdigitated electrode can use the principle of dielectrophoresis to efficiently capture cells and achieve accurate Paired together to provide ideal conditions for cell electrical fusion, and can also use electrical pulses to induce electroporation, change the permeability of cell membranes, thereby achieving cell fusion, and can use the principle of negative dielectrophoresis to keep cells away from electrodes during fusion.
  • the double-well structure of the capture hole and the culture hole can be used to transfer the fused cells to the culture hole under the influence of gravity, and then continue the subsequent culture to analyze the effect of cell fusion.
  • the present application proposes a cell electrofusion culture device.
  • the cell electrofusion culture device may include: the above cell electrofusion culture chip 100, a signal generator and a microfluidic pump; wherein the signal generator is respectively connected with at least one first interdigitated electrode 120 and at least one The second interdigitated electrode 130 is electrically connected to send different electrical signals to the cell electrofusion culture chip 100; and the microfluidic pumps are respectively connected to the two inlets and outlets 151 to control the flow of the cell suspension.
  • the present application proposes a cell electrofusion culture device, which can realize the functions of cell capture, cell pairing and cell fusion of the cell electrofusion culture device through different electrical signals of the signal generator , And the flow of cell suspension is controlled by a microfluidic pump.
  • a cell electrofusion culture device which can realize the functions of cell capture, cell pairing and cell fusion of the cell electrofusion culture device through different electrical signals of the signal generator , And the flow of cell suspension is controlled by a microfluidic pump.
  • the present application proposes a method of cell electrical fusion.
  • the method uses the above-mentioned cell electrofusion culture device, and referring to FIG. 5, the method includes:
  • the microfluidic pump is used to make the cell suspension enter the cavity from the inlet and outlet of the cell electrofusion culture chip.
  • cell feeding is performed, that is, the microfluidic pump in the cell electrofusion culture device is used to make the cell suspension enter the cavity 160 from the inlet and outlet 151 of the cell electrofusion culture chip 100.
  • the microfluidic pump in the cell electrofusion culture device is used to make the cell suspension enter the cavity 160 from the inlet and outlet 151 of the cell electrofusion culture chip 100.
  • a micro-motion table to drive a syringe that is, a micro-flow pump
  • a gravity adjustment method can be used to push it.
  • S200 A sinusoidal electrical signal is applied to the first and second interdigitated electrodes by a signal generator.
  • the cells in the cell suspension enter the trapping hole and are adsorbed to the first protruding electrode or the second protruding electrode On the electrode.
  • cell capture is performed, that is, a sinusoidal electrical signal is applied to at least one first interdigitated electrode 120 and at least one second interdigitated electrode 130 of the cell electrofusion culture chip 100 by a signal generator, which can be captured in the capture hole 141
  • a positive dielectrophoresis effect is generated between the exposed first protruding electrode 121 and the second protruding electrode 131, so that the cells in the suspension can be adsorbed to the electrode exposed at the bottom of the capture hole, that is, the one exposed in each capture hole 141
  • the first protruding electrode 121 and the second protruding electrode 131 respectively adsorb one cell.
  • R and d are the radius of cell and cell membrane respectively; with The complex permittivity of cytoplasm and cell membrane, ⁇ cyto and ⁇ ayto are the dielectric constant and conductivity of the cytoplasm, ⁇ mem and ⁇ mem are the dielectric constant and conductivity of the cell membrane, and ⁇ is the angular frequency of the electrical signal.
  • dielectrophoresis is that cells are polarized into dipoles by an external electric field in a non-uniform electric field. When the dipole is under the action of a uniform electric field, it is in a balanced state; and in a non-uniform electric field, because the electric field strength of the two-stage charge is unequal, the force applied to the two stages is unequal, at this time, The cells will be subjected to a directional migration by a net force in the direction of the greater electric field strength.
  • the formula for dielectrophoretic force is:
  • K CM is the Clausius-Mossot coefficient
  • ⁇ m is the dielectric constant of the solution
  • ⁇ m is the conductivity of the solution.
  • the relationship between Clausius-Mossot coefficient (K CM ) and frequency (Frequency) is shown in Figure 8.
  • the magnitude of the dielectrophoretic force depends on the magnitude of the electric field gradient.
  • the selected parameters make the dielectrophoretic force positive, that is, under the action of the electric field, the cells will undergo a directional migration to the area with a high electric field gradient. Therefore, as long as the electric field distribution and electric signal configuration can be accurately controlled, the cells in the electric field can be subjected to electric field forces of different sizes and directions, which in turn can control the movement of cells in certain specific areas.
  • step S200 cell pairing is performed, that is, by adjusting the signal frequency of the signal generator, the cells adsorbed by the first protruding electrode 121 and the cells adsorbed by the second protruding electrode 131 in step S200 can be contact-paired.
  • FIG. 9 is an example of a positive dielectrophoresis effect (FIG. 9 (a)) and a negative dielectrophoresis effect (FIG. 9 (b)), and the length of the scale is 15 ⁇ m.
  • S400 Apply an electrical pulse signal to the first and second interdigitated electrodes by adjusting the signal generator to cause electroporation and fusion of the contact parts of the cells.
  • cell fusion is performed, that is, by continuing to adjust the signal generator, an electrical pulse signal is applied to at least one first interdigitated electrode 120 and at least one second interdigitated electrode 130, so that the contacting part of the cell is electroporated and Fusion.
  • the cell membrane can be broken down under the action of high-voltage electrical pulses, and if the pulse width is controlled within a certain range, the cell membrane can be recovered after perforation.
  • the above-mentioned reversible electroporation process is the basic of cell fusion principle. Since the phospholipid bilayer of the cell membrane can be approximated as an insulator, and the liquid on both sides of the cell membrane (intracellular fluid and cell suspension) is an electrolyte solution, the application of an electric field outside the cell causes polarization of the electrolyte ions on both sides to induce the cell membrane V a potential difference is formed. It causes external electric breakdown of the cell membrane that can be applied to the external electric field caused by ionic polarization on both sides of the electrolyte membrane, and thus induce the formation of cell membrane potential difference V a. among them,
  • V a 1.5aEcos ⁇ / [1+ ( ⁇ ) 2 ] 1/2
  • ⁇ is the angle between the direction of the electric field and the normal of the specified point
  • ⁇ is the angular frequency of the applied AC electric field
  • ⁇ is the relaxation time constant of the cell membrane dielectric.
  • h is the thickness of the film after being subjected to an electric field
  • ⁇ e is the dielectric constant of the buffer.
  • V e is the critical voltage.
  • the film will be broken due to instability.
  • Fig. 10 shows a simulation diagram of capturing the electric field distribution in the hole at the instant of applying an electric pulse signal, with a scale of 10 microns.
  • the number of electrical pulse signals may be 5, the pulse width may be 50 microseconds, and the frequency may be 1 Hz.
  • the electrical pulse signals using the above parameters can better electroporate the cells.
  • cells are first adsorbed to the electrode by the force of dielectrophoresis, and then electrical breakdown occurs under the action of pulsed electrical signals. When the cell potential difference reaches the breakdown voltage, electroporation occurs and electrical fusion is achieved.
  • Figure 11 shows an example of the use of Heila cell fusion chip in this cell electrofusion chip, the scale is 5 microns.
  • the method may further include:
  • step S400 cell transfer is performed, that is, after the cell fusion is completed in step S400, the cell electrofusion culture chip can be turned 180 °, and the fused cells will fall into the culture hole 152 under the action of gravity.
  • the microfluidic pump is used to feed the culture medium into the culture hole of the cell electrofusion culture chip to culture the fused cells.
  • step S500 cell culture is performed, that is, after the fused cells are moved to the culture well 152 in step S500, and then the medium is passed into the cell electrofusion culture chip, the fused cells can be directly cultured in situ, which is convenient and quick.
  • Figure 12 shows an example of cell culture with a scale of 100 microns.
  • the present application proposes a cell electrofusion method, which uses the principle of positive dielectrophoresis to accurately and efficiently capture and pair cells, improve the stability and accuracy of cell pairing, and The principle of negative dielectrophoresis is used to keep the cells away from the electrodes during fusion to prevent damage to the cells caused by the electrical pulse signal during fusion.
  • a cell electrofusion method which uses the principle of positive dielectrophoresis to accurately and efficiently capture and pair cells, improve the stability and accuracy of cell pairing, and The principle of negative dielectrophoresis is used to keep the cells away from the electrodes during fusion to prevent damage to the cells caused by the electrical pulse signal during fusion.
  • first and second are used for description purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated.
  • features defined as “first” and “second” may include at least one of the features, either explicitly or implicitly.
  • the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise specifically limited.

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Abstract

提出了细胞电融合培养芯片（100）、细胞电融合培养装置和细胞电融合的方法。细胞电融合培养芯片（100）包括：基底（110）；至少一个第一叉指电极（120）和至少一个第二叉指电极（130），设置在基底（110）的上表面，且每个第一叉指电极（120）具有至少一个第一凸出电极（121），每个第二叉指电极（130）具有至少一个第二凸出电极（131）；捕获层（140），覆盖至少一个第一叉指电极（120）和至少一个第二叉指电极（130）且具有至少一个捕获孔（141），捕获孔（141）贯穿捕获层（140）；培养层（150），覆盖捕获层（140），培养层（150）的下表面与捕获层（140）的上表面之间限定有腔体（160），且培养层（150）还设置有两个进出口（151）和至少一个培养孔（152），进出口（151）贯穿培养层（150）且用于细胞悬浮液的进出，培养孔（152）不贯穿培养层（150）且与捕获孔（141）正对设置；其中，腔体（160）通过捕获孔（141）与部分的第一凸出电极（121）和部分的第二凸出电极（131）相连。

Description

细胞电融合培养芯片和装置、细胞电融合的方法 技术领域
本申请涉及细胞融合技术领域，具体的，本申请涉及细胞电融合培养芯片、细胞电融合培养装置和细胞电融合的方法。
背景技术
现阶段，多倍体癌细胞(每个染色体都有两个以上副本的细胞)比大多数其他癌细胞体积更大，对化疗和放疗具有抵抗力，并且与疾病复发有关。这些巨细胞比其他癌细胞更硬，而且能够移动得更远，这可能有助于解释它们与更严重疾病相关的原因。多倍体癌细胞通常来源于癌细胞的异常分裂，也可以通过细胞融合的方式人工制备。
细胞融合，是指在人工诱导或者自发的情况下，两个细胞或者原生质体融合形成一个细胞，在遗传物质的研究方面有着重要的意义。细胞电融合技术是通过外界电场控制两个或多个细胞融合变成一个单核或多核杂合细胞的方法。相较于生物、化学和物理等诱导融合技术而言，细胞电融合技术具有效率较高、操作简便、对细胞无毒害等优点，逐渐成为了生物工程研究领域的重要手段。
微流控芯片具有高通量、消耗试样和试剂极少等特点，因而被广泛应用于生物、化学、医学领域的分析与检测。基于微流控技术的细胞融合器件能够在消耗少量试剂的同时，短时间内实现对大量细胞的稳定融合。
操纵细胞是实现细胞融合的前提，细胞按照需要实现配对。介电泳是电介质在非匀强电场中受力的现象，常被用来操纵流体中的微小颗粒。介电泳力的存在不需要物体本身带电。所有粒子在电场环境里都存在介电泳现象，然而力的大小很大程度取决于物质和粒子的电学性质，粒子的形状和大小，以及场强变化率。因此，特定频率的电场可以有选择性地控制特定粒子。借助介电泳力，我们能够更加方便地实现对细胞的操纵。
单细胞的分析在细胞生物学的研究中越来越占据重要地位。对于细胞融合而言，如果能够追踪融合后单个融合细胞的生长情况，无疑对评价细胞融合的效果与融合细胞的研究有着重大的意义。但现有的基于微流控技术的细胞融合器件大多只能实现原位培养分析，不能有效区分不同细胞生长形成的群落，且融合细胞的生长会受到未融合细胞的干扰。基于此我们提出了一种基于电融合原理的细胞融合器件，使用介电泳力实现高通量高效率的细胞捕获与配对，并能在融合之后实现对单个融合细胞的培养分析。
发明内容
本申请是基于发明人的下列发现而完成的：
本申请人在研究过程中发现，现有的融合技术还存在以下问题：1)细胞配对的不稳定性：因细胞易受流体力等外力的影响，细胞的分布难以被精确控制。常用的细胞电融合器件都是利用细胞极化后的互相吸引作用使其相连成串。由于细胞极化的效果不一，因此在这种细胞电融合器件中难以精确控制细胞成串的数目，从而对细胞融合的效果产生较大影响。2)对细胞的不可逆伤害：常用的化学、生物等细胞融合方法融合效率较低且对细胞伤害较大。而常见的细胞电融合器件在工作时，细胞与电极直接接触，易对细胞造成损伤。3)融合结果分析的不便利性：常用的细胞融合器件需要将融合细胞取出后才能进行后续的培养分析，难以跟踪单个融合细胞的生长状况。
所以，本申请的发明人经过深入研究，设计一种结合介电泳技术和双阱结构的、能够对细胞进行高通量融合的细胞电融合培养芯片和装置，其集电融合细胞和细胞培养两种功能于一体，这两种功能进一步允许对细胞融合之后的情况进行培养分析。本申请设计的细胞电融合培养芯片可利用介电泳原理高效快速阵列化地捕获和配对细胞，使用电脉冲融合已经配对的细胞，并利用双阱结构实现对融合细胞的培养分析。
基于本申请的细胞电融合培养芯片和装置对细胞融合及其后续培养分析在操作上的高通量与集成化，本申请能够在基于细胞融合的细胞遗传研究中发挥重要作用。
在本申请的第一方面，本申请提出了一种细胞电融合培养芯片。
根据本申请的实施例，所述细胞电融合培养芯片包括：基底；至少一个第一叉指电极和至少一个第二叉指电极，所述至少一个第一叉指电极和至少一个第二叉指电极设置在所述基底的上表面，且每个所述第一叉指电极具有至少一个第一凸出电极，每个所述第二叉指电极具有至少一个第二凸出电极；捕获层，所述捕获层覆盖所述至少一个第一叉指电极和至少一个第二叉指电极，且具有至少一个捕获孔，所述捕获孔贯穿所述捕获层；培养层，所述培养层覆盖所述捕获层，所述培养层的下表面与所述捕获层的上表面之间限定有腔体，且所述培养层还设置有两个进出口和至少一个培养孔，所述进出口贯穿所述培养层且用于细胞悬浮液的进出，所述培养孔不贯穿所述培养层且与所述捕获孔正对设置；其中，所述腔体通过所述捕获孔与部分的所述第一凸出电极和部分的所述第二凸出电极相连。
发明人经过研究发现，本申请实施例的细胞电融合培养芯片，其第一叉指电极和第二叉指电极可利用介电泳原理实现高效地捕获细胞并实现精确的两两配对，从而为细胞电融合提供理想的条件，还可利用电脉冲引发电穿孔现象，改变细胞膜通透性，从而实现细胞融合，而且能利用负介电泳原理使细胞在融合时远离电极，从而防止电极对细胞造成损伤，并且能利用捕获孔和培养孔的双阱结构，使融合的细胞在重力的作用下转移到培养孔内， 进而继续后续的培养以分析细胞融合的效果。
另外，根据本申请上述实施例的细胞电融合培养芯片，还可具有如下附加的技术特征：
根据本申请的实施例，形成所述基底的材料为无机玻璃或有机玻璃，形成所述第一叉指电极和所述第二叉指电极的材料为金属或导电聚合物，形成所述捕获层的材料为光刻胶，形成所述培养层的材料包括聚二甲基硅氧烷。
根据本申请的实施例，所述第一叉指电极和所述第二叉指电极的厚度为50～150纳米，方形的所述捕获孔的长为10～100微米、宽为5～50微米，一个所述捕获孔中的所述第一凸出电极与所述第二凸出电极的间距为20～30微米。
根据本申请的实施例，所述细胞电融合培养芯片包括相间平行排布的多个所述第一叉指电极和多个所述第二叉指电极，且每个所述第一叉指电极具有多个所述第一凸出电极，每个所述第二叉指电极具有多个所述第二凸出电极；所述捕获层具有多个所述捕获孔，所述培养层具有多个所述培养孔，并且，所述多个捕获孔和所述多个培养孔都呈阵列排布。
根据本申请的实施例，所述捕获孔的个数为50～1000个。
根据本申请的实施例，所述捕获层与所述培养层之间可拆开。
在本申请的第二方面，本申请提出了一种细胞电融合培养装置。
根据本申请的实施例，所述细胞电融合培养装置包括：上述的细胞电融合培养芯片；信号发生器，所述信号发生器分别与所述至少一个第一叉指电极和至少一个第二叉指电极电连接，用于对所述细胞电融合培养芯片发出不同的电信号；微流泵，所述微流泵分别与所述两个进出口相连，用于控制细胞悬浮液的流动。
发明人经过研究发现，本申请实施例的细胞电融合培养装置，通过信号发生器的不同电信号可实现细胞电融合培养装置的细胞捕获、细胞配对和细胞融合的功能，并通过微流泵控制细胞悬浮液的流动。本领域技术人员能够理解的是，前面针对细胞电融合培养芯片所描述的特征和优点，仍适用于该细胞电融合培养装置，在此不再赘述。
在本申请的第三方面，本申请提出了一种细胞电融合的方法。
根据本申请的实施例，所述方法使用上述的细胞电融合培养装置，且所述方法包括：通过微流泵使细胞悬浮液从细胞电融合培养芯片的进出口进入腔体；通过信号发生器对第一叉指电极和第二叉指电极施加正弦电信号，所述细胞悬浮液中的细胞进入捕获孔中，并被吸附到第一凸出电极或第二凸出电极上；通过调节所述信号发生器的信号频率，使所述第一凸出电极吸附的所述细胞与所述第二凸出电极吸附的所述细胞进行接触配对；通过所述调节信号发生器对所述第一叉指电极和所述第二叉指电极施加电脉冲信号，使所述细胞 的接触部分发生电穿孔并融合。
发明人经过研究发现，采用本申请实施例的细胞电融合方法，利用正介电泳原理可精准高效地对细胞进行捕获和配对，提高细胞配对的稳定性和准确性，并利用负介电泳原理使细胞在融合时远离电极，防止融合时的电脉冲信号使电极对细胞造成损伤。本领域技术人员能够理解的是，前面针对细胞电融合培养芯片、细胞电融合培养装置所描述的特征和优点，仍适用于该细胞电融合的方法，在此不再赘述。
另外，根据本申请上述实施例的方法，还可以具有如下附加的技术特征：
根据本申请的实施例，在所述细胞进行融合的步骤之后，所述方法进一步包括：将所述细胞电融合培养芯片翻转180°，使所述融合后的细胞落入培养孔；通过所述微流泵使培养基进入所述细胞电融合培养芯片的所述培养孔，对所述融合后的细胞进行培养。
根据本申请的实施例，所述电脉冲信号的个数为5个、脉宽为50微秒且频率为1Hz。
本申请的关键点和技术效果在于：
(1)利用介电泳原理和捕获孔阵列结构有效地解决了细胞捕获和配对的效率问题；提高细胞配对的稳定性和准确性。
(2)通过捕获孔和培养孔的双阱结构，并利用重力将融合后的细胞从捕获孔转移到培养孔以实现对融合结果的培养分析。
(3)利用负介电泳原理使细胞在融合时远离电极，防止融合时的电脉冲信号使电极对细胞造成损伤；
(4)利用阵列化的结构，实现高通量的细胞融合与培养分析。
(5)在同一芯片和装置上实现细胞融合与培养分析。
附图说明
图1是本申请一个实施例的细胞电融合培养芯片的斜视结构示意图；
图2是本申请一个实施例的细胞电融合培养芯片的俯视结构示意图；
图3是本申请一个实施例的细胞电融合培养芯片的横截面结构示意图；
图4是本申请另一个实施例的细胞电融合培养芯片的实例图及其放大图：
A：一个实际制作出的细胞电融合培养芯片的照片，
B：细胞电融合培养芯片的微光阵列的局部放大照片，标尺为100微米，
C：细胞电融合培养芯片的第一叉指电极和第二叉指电极的局部放大照片，标尺为100微米，
D：细胞电融合培养芯片的培养孔阵列的局部放大照片，标尺为100微米；
图5是本申请一个实施例的细胞电融合的方法流程示意图；
图6是本申请另一个实施例的细胞电融合的方法流程示意图；
图7是本申请一个实施例的细胞的球壳模型和等效的均一球体模型；
图8是本申请一个实施例的克劳休斯-莫索提系数与频率的关系曲线；
图9是本申请一个实施例的正负介电泳效应的照片；
图10是本申请一个实施例的施加电脉冲信号的瞬间捕获孔内电场分布的仿真图；
图11是本申请一个实施例的海拉细胞电融合的过程照片
图12是本申请一个实施例的融合细胞在培养孔中培养的照片。
附图标记
100 细胞电融合培养芯片
110 基底
120 第一叉指电极
121 第一凸出电极
130 第二叉指电极
131 第二凸出电极
140 捕获层
141 捕获孔
150 培养层
151 进出口
152 培养孔
160 腔体
具体实施方式
下面详细描述本申请的实施例，本技术领域人员会理解，下面实施例旨在用于解释本申请，而不应视为对本申请的限制。除非特别说明，在下面实施例中没有明确描述具体技术或条件的，本领域技术人员可以按照本领域内的常用的技术或条件或按照产品说明书进行。
在本申请的一个方面，本申请提出了一种细胞电融合培养芯片。
根据本申请的实施例，参考图1～3，细胞电融合培养芯片100包括基底110、至少一个第一叉指电极120、至少一个第二叉指电极130、捕获层140和培养层150；其中，至少一 个第一叉指电极120和至少一个第二叉指电极130，都设置在基底110的上表面，且每个第一叉指电极120具有至少一个第一凸出电极121，每个第二叉指电极130具有至少一个第二凸出电极131；捕获层140覆盖至少一个第一叉指电极120和至少一个第二叉指电极130，捕获层140具有至少一个捕获孔141，且捕获孔141贯穿捕获层140；培养层150覆盖捕获层140，培养层150的下表面与捕获层140的上表面之间限定有腔体160，且培养层150还设置有两个进出口151和至少一个培养孔152，而进出口151贯穿培养层150且用于细胞悬浮液的进出，培养孔152不贯穿培养层150且与捕获孔141正对设置；其中，腔体160通过捕获孔141与部分的第一凸出电极121和部分的第二凸出电极131相连。
根据本申请的实施例，形成基底110的材料可以为无机玻璃或有机玻璃，形成第一叉指电极120和第二叉指电极130的材料可以为金属或导电聚合物，形成捕获层140的材料可为光刻胶，形成培养层150的材料包括聚二甲基硅氧烷(PDMS)。如此，透明材料的基底110方便观察，导电材料的电极更容易将细胞极化为偶极子，选择透明的光刻胶材料可在补货层140上光刻出捕获孔141，而复杂结构的培养层150可通过用聚二甲基硅氧烷材料倒模形成。
根据本申请的实施例，第一叉指电极120和第二叉指电极130的厚度可为50～150纳米，方形的捕获孔141的长可为10～100微米、宽可为5～50微米，一个捕获孔141中的第一凸出电极121与第二凸出电极131之间的距离可为20～30微米。如此，采用上述尺寸形成的腔体160中露出部分的第一凸出电极121与第二凸出电极131之间能形成使细胞定向迁移的介电泳效应。在一些具体事例中，第一叉指电极120和第二叉指电极130可通过溅射并光刻形成20纳米厚的铂和80纳米厚的金，如此，可使电极的导电效果更好且不易损坏。
根据本申请的实施例，细胞电融合培养芯片100可包括相间平行排布的多个第一叉指电极120和多个第二叉指电极130，且每个第一叉指电极120可具有多个第一凸出电极121，每个第二叉指电极130可具有多个第二凸出电极131；而捕获层140可具有多个捕获孔141，培养层150可具有多个培养孔152，并且，多个捕获孔141和多个培养孔152都呈阵列排布。如此，培养孔阵列中的每个培养孔可与捕获孔阵列中的每个捕获孔正对设置，且每个捕获孔中都会露出部分的第一凸出电极和第二凸出电极，且参考图4，这种大规模阵列化结构设计的捕获孔和培养孔与叉指电极阵列对应设置，从而可同时捕获和电融合大量的细胞。在本申请的一些实施例中，捕获孔的个数可为50～1000个，如此，通过一次电融合过程可获得上千个融合细胞。
根据本申请的实施例，捕获层140与培养层150之间是可拆开的，具体例如通过可逆的键合作用等组装的，如此，进行电融合前先将培养层150的下表面与捕获层140的上表面键合，等到捕获测试一批细胞后将捕获层140与培养层150拆开，进行必要的清洗、消 毒处理以后，再键合成型而用于捕获下一批细胞。
根据本申请的实施例，第一叉指电极120和第二叉指电极130的宽度尺寸可根据细胞尺寸进行匹配设计，长度则根据所需的通量大小进行设计；腔体160的高度尺寸、根据细胞尺寸进行匹配设计；捕获孔141的长度、宽度和深度都根据细胞尺寸进行匹配设计。
综上所述，根据本申请的实施例，本申请提出了一种细胞电融合培养芯片，其第一叉指电极和第二叉指电极可利用介电泳原理实现高效地捕获细胞并实现精确的两两配对，从而为细胞电融合提供理想的条件，还可利用电脉冲引发电穿孔现象，改变细胞膜通透性，从而实现细胞融合，而且能利用负介电泳原理使细胞在融合时远离电极，从而防止电极对细胞造成损伤，并且能利用捕获孔和培养孔的双阱结构，使融合的细胞在重力的作用下转移到培养孔内，进而继续后续的培养以分析细胞融合的效果。
在本申请的另一个方面，本申请提出了一种细胞电融合培养装置。
根据本申请的实施例，细胞电融合培养装置可包括：上述的细胞电融合培养芯片100、信号发生器和微流泵；其中，信号发生器分别与至少一个第一叉指电极120和至少一个第二叉指电极130电连接，用于对细胞电融合培养芯片100发出不同的电信号；而微流泵分别与两个进出口151相连，用于控制细胞悬浮液的流动。
综上所述，根据本申请的实施例，本申请提出了一种细胞电融合培养装置，通过信号发生器的不同电信号可实现细胞电融合培养装置的细胞捕获、细胞配对和细胞融合的功能，并通过微流泵控制细胞悬浮液的流动。本领域技术人员能够理解的是，前面针对细胞电融合培养芯片所描述的特征和优点，仍适用于该细胞电融合培养装置，在此不再赘述。
在本申请的另一个方面，本申请提出了一种细胞电融合的方法。根据本申请的实施例，该方法使用上述的细胞电融合培养装置，且参考图5，该方法包括：
S100：通过微流泵使细胞悬浮液从细胞电融合培养芯片的进出口进入腔体。
在该步骤中，进行细胞给进，即通过细胞电融合培养装置中的微流泵，使细胞悬浮液从细胞电融合培养芯片100的进出口151进入腔体160。根据本申请的实施例，理论上不仅可采用微动台驱动注射器(即微流泵)来推动细胞悬浮液流动，也可以采用重力调节的方式进行推动。
S200：通过信号发生器对第一叉指电极和第二叉指电极施加正弦电信号，所述细胞悬浮液中的细胞进入捕获孔中，并被吸附到第一凸出电极或第二凸出电极上。
在该步骤中，进行细胞捕获，即通过信号发生器对细胞电融合培养芯片100的至少一个第一叉指电极120和至少一个第二叉指电极130施加正弦电信号，可在捕获孔141中露 出的第一凸出电极121与第二凸出电极131之间产生正介电泳效应，可使悬浮液中的细胞被吸附到捕获孔底部露出的电极上，即每个捕获孔141中露出的第一凸出电极121与第二凸出电极131分别吸附一个细胞。
需要说明的是，细胞是由细胞膜、细胞质及多种细胞器构成的，其电学特性比较复杂。因而假设细胞内部结构均匀，进而将其等效为球壳模型，如图7所示。其中，等效后的细胞复介电常数为：
Figure PCTCN2018114845-appb-000001
式中，R和d分别为细胞和细胞膜的半径；
Figure PCTCN2018114845-appb-000002
和
Figure PCTCN2018114845-appb-000003
分别为细胞质和细胞膜的复介电常数，
Figure PCTCN2018114845-appb-000004
ε cyto和σ ayto分别为细胞质的介电常数和电导率，ε mem和σ mem分别为细胞膜的介电常数和电导率，ω为电信号的角频率。
所以，由于细胞膜具有非导电性，整个细胞可以被简化为一个非带电粒子。介电泳的实质就是细胞在非均匀电场中被外界电场极化为偶极子。当该偶极子处于均匀电场作用下，它处于平衡状态；而在非均匀的电场中，因为两级电荷处所处的电场强度不等，所以两级所受的作用力不等，此时，细胞将受到电场强度较大方向的一个净力推动细胞进行定向迁徙。介电泳力的公式为：
Figure PCTCN2018114845-appb-000005
Figure PCTCN2018114845-appb-000006
Figure PCTCN2018114845-appb-000007
其中，K CM是克劳休斯-莫索提系数，ε m是溶液的介电常数，σ m是溶液的电导率。
而克劳休斯-莫索提系数(K CM)与频率(Frequency)的关系如图8所示。介电泳力大小决定于电场梯度大小，选定参数使介电泳力为正介电泳力，即在电场作用下细胞将向电场梯度高的区域发生定向迁徙运动。因此，只要能够精准地控制电场分布与电信号配置，就可以使电场中的细胞受到不同大小和方向的电场力，进而能够控制某些特定区域细胞运动。
S300：通过调节信号发生器的信号频率，使第一凸出电极吸附的细胞与第二凸出电极捕获的细胞进行接触配对。
在该步骤中，进行细胞配对，即通过调节信号发生器的信号频率，可使步骤S200中第 一凸出电极121吸附的细胞与第二凸出电极131吸附的细胞进行接触配对。
还需要说明的是，当Re[K_CM]>0时，产生正介电泳效应，微粒被电极吸引；当Re[K_CM]<0时，产生负介电泳效应，微粒被电极排斥。所以，可以通过调节加载在电极上的信号频率，以实现正负介电泳的切换。具体的，图9为一个正介电泳效应(图9的(a))和负介电泳效应(图9的(b))的实例，其标尺的长度为15微米。
S400：通过调节信号发生器对第一叉指电极和第二叉指电极施加电脉冲信号，使细胞的接触部分发生电穿孔并融合。
在该步骤中，进行细胞融合，即继续通过调节信号发生器，对至少一个第一叉指电极120和至少一个第二叉指电极130施加电脉冲信号，可使细胞的接触部分发生电穿孔并融合。
需要说明的是，细胞膜在受高压电脉冲的作用下可以被击穿，并且如果控制脉冲宽度在一定的范围内可使细胞膜在穿孔后恢复，上述的可逆电穿孔过程即为细胞融合的基本原理。由于细胞膜的磷脂双分子层可被近似为绝缘体，而细胞膜两侧的液体(细胞内液和细胞悬浮液)都是电解质溶液，因此在细胞外施加电场引起两侧电解质离子极化即可诱导细胞膜电位差V a的形成。外界电场能够击穿细胞膜的原因在于，施加外界电场将引起细胞膜两边的电解质离子极化，进而诱导形成细胞膜电位差V a。其中，
V a＝1.5aEcosθ/[1+(ωτ) 2] 1/2
式中，θ是电场方向与指定点法线之间的夹角，ω是外加交流电场的角频率，τ是细胞膜电介质的弛豫时间常数。由公式易知细胞膜上的诱导膜电位与外加电场呈正相关。而外加电压会对细胞膜产生一个压力F：
F＝0.5s es 0(V/h) 2
式中，h是膜的受电场作用后的厚度，ε e是缓冲液的介电常数。当外界电场产生的压力施加于一块初始厚度为h 0的膜上时，膜就会受压变薄，薄厚的改变程度取决于膜的弹性常数Y：
Y ln(h/h 0)＝F＝0.5s es 0(V e/h) 2
Figure PCTCN2018114845-appb-000008
式中，V e为临界电压。通常，在ln(h/h 0)＝-1/2时，即临界厚度h＝0.607h 0时，膜就会因为不稳定而被击穿。图10展示了施加电脉冲信号的瞬间捕获孔内电场分布的仿真图，其标尺为10微米。
根据本申请的实施例，电脉冲信号的个数可为5个、脉宽可为50微秒且频率可为1Hz，如此，采用上述参数的电脉冲信号可使细胞更好地发生电穿孔。而在细胞电融合芯片中，细胞先受介电泳力的作用下吸附到电极处，然后在脉冲电信号作用下发生电击穿。当细胞电位差达到击穿电压时，会发生电穿孔，并实现电融合。图11展示了本细胞电融合芯片使用一个海拉细胞融合实例，其标尺为5微米。
在本申请的另一些实施例中，参考图6，在步骤S400之后，该方法可进一步包括：
S500：将细胞电融合培养芯片翻转180°，使融合后的细胞落入培养孔。
在该步骤中，进行细胞转移，即在步骤S400完成细胞融合之后，可将细胞电融合培养芯片翻转180°，融合细胞会在重力作用下落至培养孔152中。
S600：通过微流泵使培养基进入细胞电融合培养芯片的培养孔，对融合后的细胞进行培养。
在该步骤中，进行细胞培养，即待步骤S500中融合细胞移至培养孔152中后，再向细胞电融合培养芯片通入培养基，即可原位对融合细胞直接进行培养，方便快捷。图12展示了一个细胞培养实例，其标尺为100微米。
综上所述，根据本申请的实施例，本申请提出了一种细胞电融合方法，利用正介电泳原理可精准高效地对细胞进行捕获和配对，提高细胞配对的稳定性和准确性，并利用负介电泳原理使细胞在融合时远离电极，防止融合时的电脉冲信号对细胞造成损伤。本领域技术人员能够理解的是，前面针对细胞电融合培养芯片、细胞电融合培养装置所描述的特征和优点，仍适用于该细胞电融合的方法，在此不再赘述。
在本申请的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
在本申请的描述中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本申请中的具体含义。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者 隐含地包括至少一个该特征。在本申请的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本申请的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本申请的限制，本领域的普通技术人员在本申请的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种细胞电融合培养芯片，包括：
    基底；
    至少一个第一叉指电极和至少一个第二叉指电极，所述至少一个第一叉指电极和至少一个第二叉指电极设置在所述基底的上表面，且每个所述第一叉指电极具有至少一个第一凸出电极，每个所述第二叉指电极具有至少一个第二凸出电极；
    捕获层，所述捕获层覆盖所述至少一个第一叉指电极和至少一个第二叉指电极，且具有至少一个捕获孔，所述捕获孔贯穿所述捕获层；
    培养层，所述培养层覆盖所述捕获层，所述培养层的下表面与所述捕获层的上表面之间限定有腔体，且所述培养层还设置有两个进出口和至少一个培养孔，所述进出口贯穿所述培养层且用于细胞悬浮液的进出，所述培养孔不贯穿所述培养层且与所述捕获孔正对设置；
    其中，所述腔体通过所述捕获孔与部分的所述第一凸出电极和部分的所述第二凸出电极相连。
  2. 根据权利要求1所述的细胞电融合培养芯片，
    形成所述基底的材料为无机玻璃或有机玻璃，
    形成所述第一叉指电极和所述第二叉指电极的材料为金属或导电聚合物，
    形成所述捕获层的材料为光刻胶，
    形成所述培养层的材料包括聚二甲基硅氧烷。
  3. 根据权利要求1所述的细胞电融合培养芯片，
    所述第一叉指电极和所述第二叉指电极的厚度为50～150纳米，
    方形的所述捕获孔的长为10～100微米、宽为5～50微米，
    一个所述捕获孔中的所述第一凸出电极与所述第二凸出电极的间距为20～30微米。
  4. 根据权利要求1所述的细胞电融合培养芯片，
    所述细胞电融合培养芯片包括相间平行排布的多个所述第一叉指电极和多个所述第二叉指电极，且每个所述第一叉指电极具有多个所述第一凸出电极，每个所述第二叉指电极具有多个所述第二凸出电极；
    所述捕获层具有多个所述捕获孔，所述培养层具有多个所述培养孔，并且，所述多个捕获孔和所述多个培养孔都呈阵列排布。
  5. 根据权利要求4所述的细胞电融合培养芯片，所述捕获孔的个数为50～1000个。
  6. 根据权利要求1所述的细胞电融合培养芯片，所述捕获层与所述培养层之间可拆开。
  7. 一种细胞电融合培养装置，包括：
    权利要求1～6中任一项所述的细胞电融合培养芯片；
    信号发生器，所述信号发生器分别与所述至少一个第一叉指电极和至少一个第二叉指电极电连接，用于对所述细胞电融合培养芯片发出不同的电信号；
    微流泵，所述微流泵分别与所述两个进出口相连，用于控制细胞悬浮液的流动。
  8. 一种细胞电融合的方法，使用权利要求7所述的细胞电融合培养装置，且所述方法包括：
    通过微流泵使细胞悬浮液从细胞电融合培养芯片的进出口进入腔体；
    通过信号发生器对第一叉指电极和第二叉指电极施加正弦电信号，所述细胞悬浮液中的细胞进入捕获孔中，并被吸附到第一凸出电极或第二凸出电极上；
    通过调节所述信号发生器的信号频率，使所述第一凸出电极吸附的所述细胞与所述第二凸出电极吸附的所述细胞进行接触配对；
    通过所述调节信号发生器对所述第一叉指电极和所述第二叉指电极施加电脉冲信号，使所述细胞的接触部分发生电穿孔并融合。
  9. 根据权利要求8所述的方法，在所述细胞进行融合的步骤之后，所述方法进一步包括：
    将所述细胞电融合培养芯片翻转180°，使所述融合后的细胞落入培养孔；
    通过所述微流泵使培养基进入所述细胞电融合培养芯片的所述培养孔，对所述融合后的细胞进行培养。
  10. 根据权利要求8所述的方法，所述电脉冲信号的个数为5个、脉宽为50微秒且频率为1Hz。
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