WO2022236921A1 - 细胞转染系统及方法 - Google Patents

细胞转染系统及方法 Download PDF

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WO2022236921A1
WO2022236921A1 PCT/CN2021/100663 CN2021100663W WO2022236921A1 WO 2022236921 A1 WO2022236921 A1 WO 2022236921A1 CN 2021100663 W CN2021100663 W CN 2021100663W WO 2022236921 A1 WO2022236921 A1 WO 2022236921A1
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vibration
cells
transfection
microfluidic system
area
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French (fr)
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汪家道
马原
李轩
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • 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
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • 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

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  • the present application relates to the technical field of cell transfection, in particular to a cell transfection system and method.
  • CRISPR has become the most important gene editing method due to its specificity, high efficiency, multi-target simultaneous editing and other advantages. play an important role in. Safe and efficient means of cell transfection play a particularly important role in targeted gene editing.
  • Traditional intracellular transfection techniques include the use of liposomes or polymer nanoparticles to induce cell membrane perforation and endocytosis, and polypeptide-mediated cell membrane penetration to complete drug delivery. These methods are somewhat limited by the cell type and structure of the target molecule, and have low transfection efficiencies. Viral transfection (such as retroviral transfection, etc.) requires specific binding, which cannot achieve the versatility of cell transfection, and there are problems such as residual chemical components, which pose a safety hazard.
  • Electroporation technology can achieve high-efficiency target transfection by breaking the membrane with current, but it has irreversible damage to cells, which limits its application. Microinjection transfection technology is inefficient and cannot achieve high-throughput transfection of bulk cells. Therefore, it is of great significance to study a high-throughput cell transfection method with wide versatility, high efficiency, low damage, and low chemical component residue.
  • This application aims to solve one of the technical problems in the related art at least to a certain extent.
  • one purpose of this application is to propose a cell transfection system, which can achieve high-throughput transfection, has high transfection efficiency and stability, and is low in cost and easy to control.
  • Another object of the present application is to propose a cell transfection method.
  • an embodiment of the present application proposes a cell transfection system, including: a bottom cover integrated with a large-area nanoneedle array structure, a top cover composed of PDMS with parallel flow channels, and a power module , control module and vibration module;
  • the bottom cover plate and the top cover plate are bonded to form a microfluidic system, and the transfection reagent and the cells to be transfected pass through the large area nanoneedle array area through the parallel flow channel at a specific flux;
  • the power module is connected to the vibration module and the control module, and is used to supply power to the control module and the vibration module;
  • the control module is connected with the vibration module and the microfluidic system, and is used to issue control instructions and control the fluid velocity of the transfection reagent and the cells to be transfected, and the control instructions include vibration frequency and vibration range;
  • the vibration module is connected with the microfluidic system, and is used to vibrate according to the control instruction to drive the microfluidic system to vibrate, and the vibration of the microfluidic system makes the cells to be transfected and the The large-area nanoneedle array contacts, and the transfection reagent enters the cells to be transfected.
  • cell transfection system according to the above-mentioned embodiments of the present application may also have the following additional technical features:
  • the nanoneedle is in the shape of a tip with a diameter at the bottom greater than that at the top, the diameter at the bottom is 100nm-900nm, and the height is 100nm-3000nm.
  • the area of the large-area nanoneedle array structure is 10x10mm 2 to 100x100mm 2 .
  • the parallel flow channel of the top cover plate is a rectangular parallelepiped flow channel composed of a plurality of single-row channels, and a plurality of flow channels are included at the channel inlet and the channel outlet for flow splitting.
  • the channel height of the parallel channels is 10-100 ⁇ m
  • the length is 10 m-100 mm
  • the width of a single channel is 10-500 ⁇ m
  • the number of parallel channels is 2-200.
  • the vibration amplitude is 1 micron-1 mm, and the vibration frequency is 10 Hz-500 Hz.
  • cell transfection method according to the above-mentioned embodiments of the present application may also have the following additional technical features:
  • transfection reagents are plasmids, proteins and corresponding complexes.
  • the fluid velocity of the transfection reagent and the cells to be transfected is 1 ⁇ l/min-500 ⁇ l/min.
  • the microfluidic system includes a bottom cover plate integrated with a large area array nanoneedle array structure and a top cover plate composed of PDMS with parallel flow channels, and the bottom cover plate and the top cover plate are bonded way to form a microfluidic system.
  • Transfection reagents can be fluorescent substances, plasmids, proteins and related complexes.
  • Fig. 1 is a schematic structural diagram of a cell transfection system according to an embodiment of the present application
  • FIG. 2 is an electron micrograph of a nanoneedle structure according to an embodiment of the present application.
  • Fig. 3 is a model diagram of the cover plate structure on the PDMS microfluidic channel according to one embodiment of the present application
  • Fig. 4 is the physical picture of the cover plate structure on the PDMS microfluidic channel according to one embodiment of the present application
  • FIG. 5 is a physical diagram of a shaking table/microfluidic composite system according to an embodiment of the present application.
  • Fig. 6 is a fluorescent microscope image of the transfection result according to one embodiment of the present application.
  • Fig. 7 is the fluorescence micrograph of the result of the control group according to one embodiment of the present application.
  • Fig. 8 is a schematic flowchart of a cell transfection method according to an embodiment of the present application.
  • Fig. 1 is a schematic structural diagram of a cell transfection system according to an embodiment of the present application.
  • the cell transfection system includes: a bottom cover integrated with a large area nanoneedle array structure, a top cover composed of PDMS with parallel flow channels, a power module, a control module and a vibration module.
  • the bottom cover plate and the top cover plate are bonded to form a microfluidic system, and the transfection reagent and the cells to be transfected pass through the large area nanoneedle array area through parallel flow channels at a specific flux.
  • the power module is connected with the vibration module and the control module, and is used for supplying power to the control module and the vibration module.
  • the control module is connected with the vibration module and the microfluidic system, and is used for issuing control instructions and controlling the fluid velocity of the transfection reagent and the cells to be transfected.
  • the control instructions include vibration frequency and vibration amplitude.
  • the vibration module is connected with the microfluidic system, and is used to vibrate according to the control instructions to drive the microfluidic system to vibrate. Through the vibration of the microfluidic system, the cells to be transfected contact with the large array nanoneedle array, and the transfection reagent enters the waiting area. transfected cells.
  • the large-area nanoneedle array structure on the bottom cover plate can be obtained by using the self-assembly template method and deep reactive ion etching technology.
  • thermally induced tension gradients are used to drive micro- and nano-polystyrene particles to form large-area self-assembled structures at the liquid-air interface.
  • the colloidal dispersion of hydrophobic polystyrene particles was added dropwise into the liquid pool, and the hydrophobic polystyrene particles spread on the liquid-air interface to form a loose particle monolayer structure.
  • immerse the heating plate whose temperature is much higher than the liquid surface temperature on one side of the liquid pool at a certain speed, and form a temperature gradient from high to low along the heating plate, so the liquid-air interface forms a tension gradient (liquid-air interface) Tension decreases with increasing temperature), thereby inducing the Marangoni effect.
  • the loose particles at the liquid-air interface quickly aggregate to the side away from the heating plate and form a densely arranged large-area self-assembled structure of polystyrene particles at the liquid-air interface.
  • the above-mentioned large-area self-assembled structure at the liquid-air interface is transferred to the surface of the silicon substrate by means of oblique fishing.
  • Oxygen reactive ion etching technology is used to adjust the diameter of the particles in the large-area self-assembled structure to an appropriate size to form a non-closely arranged periodic micro-nano particle structure.
  • the gold-plated layer was prepared on the surface of non-closely arranged periodic grain structure by electron beam evaporation. The particles are then removed by ultrasonic cleaning to form a periodic surface with alternately arranged gold-plated layers and silicon substrates. The substrate is placed in a hydrofluoric acid/hydrogen peroxide mixed solution for gold-catalyzed etching to form a nano-column structure. Finally, the nano-pillar structure was non-uniformly etched by sulfur hexafluoride reactive ion etching technology to form a large array nano-needle structure.
  • the diameter of the hydrophobic micro- and nano-polystyrene particles is 100nm-50 ⁇ m, and the size of the liquid pool is 4cm 2 -1m 2 .
  • the liquid in the liquid pool is deionized water, and its depth is 1mm-10cm.
  • the temperature of the heating plate is 40°C-100°C, and its power is 5w-200w.
  • the preparation area of the large-area micro-nano particle self-assembled structure is 1cm 2 -1m 2 .
  • the power of the oxygen reactive ion etching technology is 20w-100w, the etching time is 30s-10min, and the diameter of the particles after the reaction is 95%-5% of the original diameter.
  • the gold plating speed of the electron beam is The thickness of the gold layer is 1nm-100nm.
  • the solvent during ultrasonic cleaning is an organic solvent, which can be toluene, xylene, etc.
  • the ratio of hydrofluoric acid to hydrogen peroxide in the hydrofluoric acid/hydrogen peroxide mixed solution is 20:1 to 2:1, the etching time is 10min-60min, and the height of the formed nano-column is 200nm-3 ⁇ m.
  • the power of sulfur hexafluoride reactive ion etching technology is 20w-200w, and the etching time is 3s-5min.
  • a large area nanoneedle array required by the embodiment of the present application can be prepared by the above method.
  • the above method is only used as an example, and is not intended as a specific limitation of the embodiment of the present application.
  • the large surface array nanoneedle array prepared by other methods Area array nanoneedle arrays can also be applied in the embodiments of the present application.
  • the nanoneedle array structure of the large area array is integrated in the bottom cover plate of the microchannel system.
  • the bottom cover plate is composed of a silicon wafer
  • the top cover plate of the microchannel system is made of PDMS (polyethylene oxide) containing parallel channels.
  • Methylsiloxane, Polydimethylsiloxane using the bonding method to combine the upper and lower cover plates to form a microfluidic system.
  • the transfection reagent and the required transfected cells pass through the large area nanoneedle area through the parallel flow channel at a specific flux, and the puncture and detachment process of the cell and the nanoneedle array structure is realized by applying vibrations of a specific frequency and amplitude.
  • the dyeing reagent enters the cell through the cell membrane formed by the above process, thereby realizing high-throughput transfection.
  • the vibration frequency and amplitude of the vibration module can be controlled by the control module, as well as the fluid velocity of the transfection reagent and the cells to be transfected in the parallel channel in the microfluidic system.
  • the nanoneedle structure is in the shape of a needle tip whose base diameter is much larger than the top diameter, the base diameter is 100nm-900nm, and the height is 100nm-3000nm.
  • the bottom cover plate is composed of a silicon wafer containing a large area nanoneedle structure, wherein the coverage area of the large area nanoneedle structure is 10x10mm 2 to 100x100mm 2 .
  • the parallel flow channel of the top cover is a rectangular parallelepiped flow channel composed of multiple single-row channels, and multiple flow channels are included at the flow channel inlet and the flow channel outlet for flow splitting.
  • the transfection reagents are plasmids, proteins and corresponding complexes.
  • the vibration amplitude is 1 micron-1 mm.
  • the vibration frequency is 10 Hz-500 Hz.
  • the fluid rate is from 1 microliter/minute to 500 microliter/minute.
  • the PDMS microchannel is prepared by photolithographic inverted molding technology, in which the height of the microchannel is 30um, the length is 10mm (here refers to the length of the parallel cuboid channel in the middle area), the width of a single channel is 150um, the number of parallel channels is 32, and the inlet and outlet Contains a symmetrical subdivided channel structure to fully and evenly disperse cells, as shown in the model diagram in Figure 3 and the physical diagram in Figure 4.
  • the bottom silicon cover plate containing the large-area nanoneedle structure was combined with the top PDMS cover plate containing the parallel microchannel structure by bonding, and placed on a vibration table to form an overall system, as shown in Figure 5. Show.
  • the cell transfection system of the embodiment of the present application is a high-throughput physical puncture cell transfection system, which can solve the problem of preparing a large number of immune cells in cancer immunotherapy, and has high throughput, high cell activity, and high sensitivity to transfection content. There are no requirements for substances, and the transfection speed is fast.
  • vibration-assisted large-area microneedle structure piercing the cell membrane by adjusting the size and vibration parameters of the microneedle, combined with the design of the microfluidic channel, it has important theoretical and practical significance for laboratory cell transfection methods and clinical applications.
  • Figure 8 is a flow chart of a cell transfection method according to one embodiment of the present application.
  • the cell transfection method comprises the following steps:
  • Step S1 setting the vibration frequency and vibration amplitude.
  • Step S2 passing the transfection reagent and the cells to be transfected through the parallel channel on the top cover of the microfluidic system through the large area nanoneedle array area on the bottom cover of the microfluidic system at a specific flux.
  • Step S3 vibrating the microfluidic system according to the vibration frequency and vibration amplitude to realize the puncture and desorption process between the cells to be transfected and the nanoneedle array structure, and the transfection reagent enters the cells to be transfected through the formed cell membrane perforation.
  • the transfection reagents are plasmids, proteins and corresponding complexes.
  • the fluid velocity of the transfection reagent and the cells to be transfected is 1 ⁇ l/min-500 ⁇ l/min.
  • the microfluidic system includes a bottom cover integrated with a large-area nanoneedle array structure and a top cover composed of PDMS with parallel flow channels.
  • the bottom cover and the top cover form a microfluidic system by bonding. system.
  • a vibration-assisted large-area microneedle structure is proposed to penetrate the cell membrane.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

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Abstract

一种细胞转染系统及方法，该系统包括：集成有大面阵纳米针阵列结构的底部盖板、由并行流道的PDMS组成的顶部盖板；底部盖板和顶部盖板通过键合方式形成微流控系统，转染试剂与待转染细胞以特定通量经并行流道通过大面阵纳米针阵列区域；电源模块与振动模块和控制模块连接，用于为细胞转染系统供电；控制模块与振动模块和微流控系统连接，用于发出控制指令和控制转染试剂与待转染细胞的流体速度；振动模块与微流控系统连接，用于根据控制指令进行振动，带动微流控系统振动，使得待转染细胞与大面阵纳米针阵列接触，转染试剂进入待转染细胞。

Description

细胞转染系统及方法
相关申请的交叉引用
本申请要求清华大学于2021年5月12日提交的、发明名称为“细胞转染系统及方法”的、中国专利申请号“202110517427.9”的优先权。
技术领域
本申请涉及细胞转染技术领域，特别涉及一种细胞转染系统及方法。
背景技术
定向基因编辑技术CRISPR由于其特异性、高效、多目标同时编辑等优势，已经成为最主要的基因编辑方法，近些年来广泛用于各种生物研究和临床应用的探索中并在解决癌症免疫治疗中发挥重要作用。安全高效的细胞转染手段在定向基因编辑中起到尤为重要的作用。传统的细胞内转染技术包括利用脂质体或聚合物纳米颗粒诱导细胞膜穿孔以及内吞作用，多肽介导细胞膜渗透完成给药等。这些方法一定程度上受限于细胞类型和目标分子的结构，并且转染效率较低。病毒转染(例如逆转录病毒转染等)需要特异性结合，无法实现细胞转染的通用性，并且存在化学成分残留等问题，存在安全隐患。电穿孔技术通过电流破膜可以实现高效的目标转染，然而其对细胞存在不可逆损伤，因而限制了其应用。显微注射转染技术效率较低，无法实现批量细胞的高通量转染。因此，研究一种具有广泛通用性、高效率，低损伤、低化学成分残留的高通量细胞转染方法具有重要意义。
发明内容
本申请旨在至少在一定程度上解决相关技术中的技术问题之一。
为此，本申请的一个目的在于提出一种细胞转染系统，该系统可以实现高通量转染，具有较高的转染效率和稳定性，且成本低，易于控制。
本申请的另一个目的在于提出一种细胞转染方法。
为达到上述目的，本申请一方面实施例提出了一种细胞转染系统，包括：集成有大面阵纳米针阵列结构的底部盖板、由并行流道的PDMS组成的顶部盖板、电源模块、控制模块和振动模块；
所述底部盖板和所述顶部盖板通过键合方式形成微流控系统，转染试剂与待转染细胞以特定通量经所述并行流道通过所述大面阵纳米针阵列区域；
所述电源模块与所述振动模块和所述控制模块连接，用于为所述控制模块和所述振动模块供电；
所述控制模块与所述振动模块和所述微流控系统连接，用于发出控制指令和控制所述转染试剂与所述待转染细胞的流体速度，所述控制指令包括振动频率和振动幅度；
所述振动模块与所述微流控系统连接，用于根据所述控制指令进行振动，带动所述微流控系统振动，通过所述微流控系统的振动使得所述待转染细胞与所述大面阵纳米针阵列接触，所述转染试剂进入所述待转染细胞。
另外，根据本申请上述实施例的细胞转染系统还可以具有以下附加的技术特征：
进一步地，所述纳米针为底部直径大于顶端直径的针尖形状，底部直径为100nm-900nm，高度为100nm-3000nm。
进一步地，所述大面阵纳米针阵列结构的面积为10x10mm 2到100x100mm 2。
进一步地，所述顶部盖板的并行流道为多条单行流道组成的长方体流道，在流道入口和流道出口包括多条流道用于进行分流。
进一步地，所述并行流道的流道高度为10-100μm，长度为10m-100mm，单一流道宽度为10-500μm，并行流道数量为2-200条。
进一步地，所述振动幅度为1微米-1毫米，所述振动频率为10赫兹-500赫兹。
为达到上述目的，本申请另一方面实施例提出了一种细胞转染方法，包括以下步骤：
设定振动频率和振动幅度；
将转染试剂与待转染细胞以特定通量经微流控系统的顶部盖板上的并行流道通过所述微流控系统的底部盖板上的大面阵纳米针阵列区域；
根据所述振动频率和所述振动幅度对所述微流控系统进行振动，实现所述待转染细胞与纳米针阵列结构的穿刺及脱附过程，所述转染试剂通过形成的细胞膜穿孔进入所述待转染细胞。
另外，根据本申请上述实施例的细胞转染方法还可以具有以下附加的技术特征：
进一步地，所述转染试剂为质粒、蛋白及相应复合体。
进一步地，所述转染试剂与所述待转染细胞的流体速度为1微升/分钟-500微升/分钟。
进一步地，所述微流控系统包括集成有大面阵纳米针阵列结构的底部盖板和由并行流道的PDMS组成的顶部盖板，所述底部盖板和所述顶部盖板通过键合方式形成微流控系统。
本申请实施例的细胞转染系统及方法的有益效果为：
1、具有高通量，转染效率高，稳定性高，成本低，易于控制等优点，其中通量可达10L/h，是现有转染技术的数十倍。
2、对不同类型的细胞和转染试剂有广泛的适用性，转染试剂可以是荧光物质、质粒、 蛋白及相关复合物。
3、不会对细胞造成不可逆损伤。
本申请附加的方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本申请的实践了解到。
附图说明
本申请上述的和/或附加的方面和优点从下面结合附图对实施例的描述中将变得明显和容易理解，其中：
图1为根据本申请一个实施例的细胞转染系统结构示意图；
图2为根据本申请一个实施例的纳米针结构的电镜图；
图3为根据本申请一个实施例的PDMS微流道上盖板结构的模型图；
图4为根据本申请一个实施例的PDMS微流道上盖板结构的实物图；
图5为根据本申请一个实施例的振动台/微流控复合系统实物图；
图6为根据本申请一个实施例的转染结果荧光显微镜图；
图7为根据本申请一个实施例的对照组结果荧光显微镜图；
图8为根据本申请一个实施例的细胞转染方法流程示意图。
具体实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制。
下面参照附图描述根据本申请实施例提出的细胞转染系统及方法。
首先将参照附图描述根据本申请实施例提出的细胞转染系统。
图1为根据本申请一个实施例的细胞转染系统结构示意图。
如图1所示，该细胞转染系统包括：集成有大面阵纳米针阵列结构的底部盖板、由并行流道的PDMS组成的顶部盖板、电源模块、控制模块和振动模块。
底部盖板和顶部盖板通过键合方式形成微流控系统，转染试剂与待转染细胞以特定通量经并行流道通过大面阵纳米针阵列区域。
电源模块与振动模块和控制模块连接，用于为控制模块和振动模块供电。
控制模块与振动模块和微流控系统连接，用于发出控制指令和控制转染试剂与待转染细胞的流体速度，控制指令包括振动频率和振动幅度。
振动模块与微流控系统连接，用于根据控制指令进行振动，带动微流控系统振动，通过微流控系统的振动使得待转染细胞与大面阵纳米针阵列接触，转染试剂进入待转 染细胞。
进一步地，在本申请的一个实施例中，底部盖板上的大面阵纳米针阵列结构可以利用自组装模板法及深反应离子刻蚀技术获得。
具体地，作为一种制备示例，利用热诱导张力梯度驱动微、纳米聚苯乙烯颗粒在液气界面形成大面积自组装结构。首先将疏水聚苯乙烯颗粒胶体分散液滴加至液池中，疏水聚苯乙烯颗粒在液气界面铺展形成松散的颗粒单层结构。随后将温度远高于液面温度的加热板以一定的速度浸没于液池一侧，沿加热板由近及远形成由高到低的温度梯度，因此液气界面形成张力梯度(液气界面张力随温度的升高而降低)，从而诱发马兰戈尼效应。液气界面松散的颗粒在马兰戈尼效应的带动下(流体曳力)向远离加热板一侧快速聚集并形成密集排列的液气界面聚苯乙烯颗粒大面积自组装结构。最后通过倾斜捞取的方式将上述液气界面大面积自组装结构转移至硅基板表面。
利用氧气反应离子刻蚀技术对大面积自组装结构中的颗粒直径调整至合适大小，以形成非紧密排列的周期性微、纳米颗粒结构。利用电子束蒸镀在非紧密排列的周期性颗粒结构表面制备镀金层。随后利用超声清洗的方式去除颗粒以形成镀金层、硅基底交替排布的周期性表面。将基板放置于氢氟酸/双氧水混合溶液中进行金催化刻蚀以形成纳米柱结构。最后利用六氟化硫反应离子刻蚀技术对纳米柱结构进行非均匀刻蚀以形成大面阵纳米针结构。
其中，疏水微、纳米聚苯乙烯颗粒直径为100nm-50μm，液池尺寸为4cm 2-1m 2。液池中的液体为去离子水，其深度为1mm-10cm。加热板温度为40℃-100℃，其功率为5w-200w。大面积微、纳米颗粒自组装结构的制备面积为1cm 2-1m 2。氧气反应离子刻蚀技术功率为20w-100w，刻蚀时间为30s-10min，反应后颗粒的直径为原始直径的95％-5％。电子束的镀金速度为
Figure PCTCN2021100663-appb-000001
金层厚度为1nm-100nm。超声清洗时的溶剂为有机溶剂，可以是甲苯，二甲苯等。氢氟酸/双氧水混合溶液中氢氟酸与双氧水比例为20：1至2：1，刻蚀时间为10min-60min，形成纳米柱高度为200nm-3μm。六氟化硫反应离子刻蚀技术功率为20w-200w，刻蚀时间为3s-5min。
可以理解的是，通过上述方法可以制备一种本申请实施例所需的大面阵纳米针阵列，上述方法仅作为一种可以示例，不作为本申请实施例的具体限定，其他方法制备的大面阵纳米针阵列，也可以应用在本申请的实施例中。
将大面阵纳米针阵列结构集成在微流道系统的底部盖板中，作为一种示例，底部盖板由硅片组成，微流道系统顶部盖板由含有并行流道的PDMS(聚二甲基硅氧烷，Polydimethylsiloxane)组成，利用键合方式将上下盖板结合形成微流控系统。其中转染试剂与所需转染细胞并以特定通量经由并行流道通过大面阵纳米针区域，通过施加特定频率与振幅的振动实现细胞与纳米针阵列结构的穿刺及脱附过程，转染试剂通过上述过程形成的细胞膜穿孔进入细胞，从而实现高通量转染。
在本申请的实施例中，可以通过控制模块控制振动模块的振动频率和振动幅度，以及控制微流控系统中转染试剂与待转染细胞在并行流道中的流体速度。
可选地，纳米针结构为底部直径远大于顶端直径的针尖形状，底部直径为100nm-900nm，高度为100nm-3000nm。
可选地，底部盖板由含有大面阵纳米针结构的硅片组成，其中大面阵纳米针结构的覆盖面积为10x10mm 2到100x100mm 2。
可选地，顶部盖板的并行流道为多条单行流道组成的长方体流道，在流道入口和流道出口包括多条流道用于进行分流。
可选地，PDMS顶部盖板的并行流道为长方体流道，其中流道高度为10-100μm，长度为10m-100mm，单一流道宽度为10-500μm，PDMS顶部盖板的并行流道数量为2-200条。
可选地，转染试剂为质粒、蛋白及相应复合体。
可选地，振动幅度为1微米-1毫米。
可选地，振动频率为10赫兹-500赫兹。
可选地，流体速度为1微升/分钟-500微升/分钟。
下面通过一个具体实施例对本申请的细胞转染系统进行说明。
以硅片底部盖板上制备直径约300nm，高度约700nm，底部直径远大于顶端直径的大面阵纳米针阵列，如图2的电镜图所示。
利用光刻倒模技术制备PDMS微流道，其中微流道高度30um，长度10mm(此处指中间区域并行长方体流道长度)，单一流道宽度150um，并行数量为32条，且入口及出口处含有对称的细分流道结构以充分均匀分散细胞，如图3的模型图，图4的实物图所示。
利用键合的方式将含有大面阵纳米针结构的底部硅盖板与含有并行微流道结构的顶部PDMS盖板结合，并将其放置于振动台上形成整体系统，如图5实物图所示。
利用k562细胞进行验证实验，利用dextran-fitc荧光染挤作为转染试剂以验证转染试剂是否能够进入细胞内实现转染。以50微升/分钟的速度通入细胞/染剂混合溶液。振动台以80hz频率，100微米振幅运行。
利用荧光显微镜观察细胞荧光状态，实验组出现明显的荧光细胞表明大面阵纳米针在振动辅助下已经将细胞膜刺破荧光物质进入细胞内，如图6的荧光显微镜图所示。同时，对照实验组(细胞/荧光染剂混合溶液不放入振动穿刺系统内)几乎不可见带有荧光的细胞表明未经穿刺，荧光物质不能大量进入细胞，如图7的荧光显微镜图所示。
通过本申请的细胞转染系统的细胞转染方法，能够实现高通量细胞转染。
本申请实施例的细胞转染系统，是一种高通量物理穿刺的细胞转染系统，可以解决癌症免疫治疗中大量免疫细胞的制备问题，具有高通量、细胞高活性、对转染内容 物无要求、转染速度快等优势。通过振动辅助大面积微针结构穿刺细胞膜的手段，通过调整微针尺寸、振动参数，结合微流道设计，对实验室细胞转染方法及临床应用中有着重要的理论和实际意义。
其次参照附图描述根据本申请实施例提出的细胞转染方法。
图8为根据本申请一个实施例的细胞转染方法流程图
如图8所示，该细胞转染方法包括以下步骤：
步骤S1，设定振动频率和振动幅度。
步骤S2，将转染试剂与待转染细胞以特定通量经微流控系统的顶部盖板上的并行流道通过微流控系统的底部盖板上的大面阵纳米针阵列区域。
步骤S3，根据振动频率和振动幅度对微流控系统进行振动，实现待转染细胞与纳米针阵列结构的穿刺及脱附过程，转染试剂通过形成的细胞膜穿孔进入待转染细胞。
可选地，转染试剂为质粒、蛋白及相应复合体。
可选地，转染试剂与待转染细胞的流体速度为1微升/分钟-500微升/分钟。
可选地，微流控系统包括集成有大面阵纳米针阵列结构的底部盖板和由并行流道的PDMS组成的顶部盖板，底部盖板和顶部盖板通过键合方式形成微流控系统。
需要说明的是，前述对系统实施例的解释说明也适用于该实施例的方法，此处不再赘述。
根据本申请实施例提出的细胞转染方法，提出了一种振动辅助大面积微针结构穿刺细胞膜的手段，通过调整微针尺寸、振动参数，结合微流道设计，可以实现高通量的细胞转染，对实验室细胞转染方法及临床应用中有着重要的理论和实际意义。
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. 一种细胞转染系统，其特征在于，包括：集成有大面阵纳米针阵列结构的底部盖板、由并行流道的PDMS组成的顶部盖板、电源模块、控制模块和振动模块；
    所述底部盖板和所述顶部盖板通过键合方式形成微流控系统，转染试剂与待转染细胞以特定通量经所述并行流道通过所述大面阵纳米针阵列区域；
    所述电源模块与所述振动模块和所述控制模块连接，用于为所述控制模块和所述振动模块供电；
    所述控制模块与所述振动模块和所述微流控系统连接，用于发出控制指令和控制所述转染试剂与所述待转染细胞的流体速度，所述控制指令包括振动频率和振动幅度；
    所述振动模块与所述微流控系统连接，用于根据所述控制指令进行振动，带动所述微流控系统振动，通过所述微流控系统的振动使得所述待转染细胞与所述大面阵纳米针阵列接触，所述转染试剂进入所述待转染细胞。
  2. 根据权利要求1所述的系统，其特征在于，所述纳米针为底部直径大于顶端直径的针尖形状，底部直径为100nm-900nm，高度为100nm-3000nm。
  3. 根据权利要求1所述的系统，其特征在于，所述大面阵纳米针阵列结构的面积为10x10mm 2到100x100mm 2。
  4. 根据权利要求1所述的系统，其特征在于，所述顶部盖板的并行流道为多条单行流道组成的长方体流道，在流道入口和流道出口包括多条流道用于进行分流。
  5. 根据权利要求1所述的系统，其特征在于，所述并行流道的流道高度为10-100μm，长度为10m-100mm，单一流道宽度为10-500μm，并行流道数量为2-200条。
  6. 根据权利要求1所述的系统，其特征在于，所述振动幅度为1微米-1毫米，所述振动频率为10赫兹-500赫兹。
  7. 一种细胞转染方法，其特征在于，包括以下步骤：
    设定振动频率和振动幅度；
    将转染试剂与待转染细胞以特定通量经微流控系统的顶部盖板上的并行流道通过所述微流控系统的底部盖板上的大面阵纳米针阵列区域；
    根据所述振动频率和所述振动幅度对所述微流控系统进行振动，实现所述待转染细胞与纳米针阵列结构的穿刺及脱附过程，所述转染试剂通过形成的细胞膜穿孔进入所述待转染细胞。
  8. 根据权利要求7所述的方法，其特征在于，所述转染试剂为质粒、蛋白及相应复合体。
  9. 根据权利要求7所述的方法，其特征在于，所述转染试剂与所述待转染细胞的流体速度为1微升/分钟-500微升/分钟。
  10. 根据权利要求7所述的方法，其特征在于，所述微流控系统包括集成有大面阵纳米针阵列结构的底部盖板和由并行流道的PDMS组成的顶部盖板，所述底部盖板和所述顶部盖板通过键合方式形成微流控系统。
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DAISUKE MATSUMOTO, RAMACHANDRA RAO SATHULURI, YOSHIO KATO, YARON R. SILBERBERG, RYUZO KAWAMURA, FUTOSHI IWATA, TAKESHI KOBAYASHI, : "Oscillating high-aspect-ratio monolithic silicon nanoneedle array enables efficient delivery of functional bio-macromolecules into living cells", SCIENTIFIC REPORTS, vol. 5, pages 15325, XP055352589, DOI: 10.1038/srep15325 *
HUANG DONG, ZHAO DEYAO, LI JINHUI, WU YUTING, DU LILI, XIA XIN-HUA, LI XIAOQIONG, DENG YULIN, LI ZHIHONG, HUANG YUANYU: "Continuous Vector-free Gene Transfer with a Novel Microfluidic Chip and Nanoneedle Array", CURRENT DRUG DELIVERY, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 16, no. 2, 14 December 2018 (2018-12-14), NL , pages 164 - 170, XP093003071, ISSN: 1567-2018, DOI: 10.2174/1567201815666181017095044 *

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