WO2020048141A1 - High-throughput microfluidic chip, and preparation method and use thereof - Google Patents

High-throughput microfluidic chip, and preparation method and use thereof Download PDF

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WO2020048141A1
WO2020048141A1 PCT/CN2019/083649 CN2019083649W WO2020048141A1 WO 2020048141 A1 WO2020048141 A1 WO 2020048141A1 CN 2019083649 W CN2019083649 W CN 2019083649W WO 2020048141 A1 WO2020048141 A1 WO 2020048141A1
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microfluidic chip
cell
throughput
recognition
chip according
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Chinese (zh)
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蒋兴宇
刘晓艳
郑文富
杨戈
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国家纳米科学中心
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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
    • C12M23/34—Internal compartments or partitions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

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  • the invention belongs to the field of medicinal chemistry, and particularly relates to a high-throughput microfluidic chip, and a preparation method and application thereof.
  • High-throughput drug screening refers to the use of microplates as a screening tool at the molecular or cell level to detect a large number of samples with rapid and sensitive automated detection methods. Therefore, it is of great significance for the discovery and evaluation of new drugs.
  • high-throughput drug screening is mainly achieved through microplates. Although it solves the disadvantages of long time and high cost of traditional drug screening methods, there are still some problems that need to be solved urgently: Drug screening based on microplates is due to the cell culture platform. Too simple to simulate cell-cell and cell-microenvironment interactions in animals and even humans.
  • the microfluidic chip Compared with microplates, the microfluidic chip requires fewer cells to operate. Because of its micrometer-scale space and relatively independent, closed environment, it is widely used in drug screening and other fields. At present, the drug screening at the microfluidic chip level is mainly through the following methods: (1) High-throughput screening based on perfusion mode. (2) High-throughput screening based on microdroplets. (3) High-throughput screening in microarray mode. Although the above technical methods can improve the throughput of drug screening, for the study of cell-cell and cell-microenvironment interactions, the design of these microfluidic chips still cannot achieve precise control and manipulation of the cell microenvironment.
  • the existing microfluidic chip-based drug screening mainly reads out signals such as fluorescence intensity, and the image analysis parameters are single and low speed. Therefore, current drug screening platforms based on microfluidic cells still have significant limitations in cell-microenvironment mutual recognition and image analysis.
  • microfluidic chip that can be used for co-culture of neurons and glial cells to study the effect of glial cells on the transfection efficiency of neurons.
  • this microfluidic chip cannot meet the high-throughput needs of drug screening.
  • traditional microfluidic chip-based drug screening usually uses fluorescence signals for characterization of drug metabolism and cytotoxicity, or the chip is used in combination with an ESI-Q-TOF mass spectrometer.
  • this kind of microfluidic chip and mass spectrometry screening platform can achieve the purpose of drug screening, the use of mass spectrometry for drug metabolism evaluation has great limitations.
  • mass spectrometry analysis methods are complicated, especially for protein analysis. Mass spectrometric analysis is cumbersome and has a narrow application range.
  • the existing methods have single image analysis parameters, low speed and difficult to quantify.
  • the purpose of the present invention is to overcome the defects in the prior art, and provide a high-throughput microfluidic chip, and a preparation method and application thereof.
  • PDMS means: polydimethylsiloxane.
  • a first aspect of the present invention provides a high-throughput microfluidic chip, the microfluidic chip includes a central nerve cell chamber and a cancer cell chamber surrounding the neural cell chamber, The middle of the nerve cell compartment and the cancer cell compartment is connected by a microchannel;
  • the microfluidic chip includes two to six cancer cell compartments, and most preferably four.
  • microfluidic chip according to the first aspect of the present invention, wherein the length of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
  • the width of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm; and / or
  • the thickness of the nerve cell compartment is 10 to 200 ⁇ m, preferably 50 to 150 ⁇ m, and most preferably 100 ⁇ m.
  • microfluidic chip according to the first aspect of the present invention, wherein the length of the cancer cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
  • the width of the cancer cell compartment is 1 to 10 mm, preferably 1 to 3 mm, and most preferably 2 mm; and / or
  • the thickness of the cancer cell compartment is 10 to 200 ⁇ m, preferably 50 to 150 ⁇ m, and most preferably 100 ⁇ m.
  • microfluidic chip according to the first aspect of the present invention, wherein the length of the microchannel is 100 to 500 ⁇ m, preferably 100 to 300 ⁇ m, and most preferably 200 ⁇ m;
  • the width of the microchannel is 5-50 ⁇ m, preferably 5-20 ⁇ m, and most preferably 10 ⁇ m; and / or
  • the thickness of the microchannel is 5 to 50 ⁇ m, preferably 5 to 20 ⁇ m, and most preferably 10 ⁇ m.
  • a second aspect of the present invention provides a method for preparing a microfluidic chip according to the first aspect, the method including the following steps:
  • the mass ratio of PDMS to the cross-linking agent in the PDMS prepolymer is 5 to 20: 1, preferably 5 to 15: 1 , Most preferably 10: 1.
  • the heating temperature is 50 to 150 ° C, preferably 50 to 100 ° C, and most preferably 80 ° C.
  • a third aspect of the present invention provides a high-throughput drug screening platform, which includes the microfluidic chip and high-content imaging system described in the first aspect.
  • the fourth aspect of the present invention provides the microfluidic chip according to the first aspect or the high-throughput drug screening platform according to the third aspect, in preparing a drug screening product, especially a high-throughput drug screening product. Application.
  • the drug is a neuro-tumor microenvironment-related drug.
  • a fifth aspect of the present invention provides a method for screening a drug, the method using:
  • microfluidic chip according to the first aspect; and / or
  • a sixth aspect of the present invention provides a cell co-culture device, the device comprising the microfluidic chip described in the first aspect.
  • a seventh aspect of the present invention provides a cell co-culture method, which uses the microfluidic chip described in the first aspect.
  • An eighth aspect of the present invention provides a cell recognition platform including the microfluidic chip described in the first aspect
  • the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
  • a ninth aspect of the present invention provides a cell identification method, which uses the microfluidic chip described in the first aspect
  • the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
  • the object of the present invention is based on a microfluidic chip with high-throughput characteristics.
  • a high-content imaging system and a special computer image analysis algorithm are used to detect cancer cells and cells.
  • the interactions between neurites were analyzed in batches in order to successfully screen anticancer drugs related to the neuro-tumor microenvironment.
  • cytokines secrete nerve growth factors (such as NGF, etc.) and neurotrophic factors (such as BDNF, GAL, NT-3, etc.).
  • nerve growth factors such as NGF, etc.
  • neurotrophic factors such as BDNF, GAL, NT-3, etc.
  • the expression of these cytokines is closely related to the formation of the neuro-tumor microenvironment.
  • the invention is based on a microfluidic chip with microchannels, as a platform for mutual recognition of cancer cells and neurites, and combined with a high-content imaging analysis system and a unique image analysis algorithm, high-throughput screening of different siRNAs.
  • the microfluidic chip in the present invention not only has microchannels that can provide directional growth of neurites, but also can realize co-culture of different cells.
  • this chip has five chambers. Neurons are cultured in the central chamber, and different chambers are screened for different drugs. Combined with a 12-well plate, 48 different drugs can be realized at one time, which has the characteristics of high throughput.
  • the microfluidic chip in the present invention can realize precise control of neurite outgrowth, and the design of different chambers can realize co-culture of nerves and cancer cells.
  • this microfluidic chip has high-throughput characteristics, and it matches Invitro Scientific's 12-well plate, which can screen 48 different drugs at one time.
  • the unique image analysis algorithm used in the present invention can evaluate the morphology of mutual recognition between neurites during cancer cell migration.
  • the microfluidic chip of the present invention is used as a high-throughput analysis platform for cell co-culture, and combined with high-content cells, an in vitro high-throughput drug screening platform is established, and automatic real-time imaging observation of the cells is realized to realize the convenience of operation
  • the present invention uses a unique image analysis algorithm to meet all the needs of drug development.
  • the high-throughput microfluidic chip of the present invention can have, but is not limited to, the following beneficial effects:
  • the invention relates to the field of high-throughput drug screening using a microfluidic chip combined with a high-content imaging system, and can be used for screening antitumor drugs (including small molecule anticancer drugs, siRNA, CRISPR / Cas9) related to the neuro-tumor microenvironment. System, etc.), and can evaluate the anti-tumor effect of the same drug in different types of cancer cell systems.
  • This high-throughput microfluidic chip-based screening system for inter-cell recognition imaging makes the speed of drug screening greatly improved. Saves time and costs.
  • the invention has broad application prospects in the field of high-throughput drug screening.
  • the image analysis algorithm applied in the present invention is of great significance for studying the mechanism of cell-cell interaction in the tumor microenvironment.
  • FIG. 1 shows a schematic diagram of a high-throughput microfluidic chip of the present invention.
  • FIG. 2 shows the co-culture of nerves and cancer cells in different chambers of the high-throughput microfluidic chip of the present invention in Experimental Example 1.
  • FIG. 2a shows a physical picture of the high-throughput microfluidic chip of the present invention. The size of the chip can just fit into a 12-well plate.
  • Figure 2b shows co-culture of hippocampal neurons and glioma cells, and
  • Figure 2c shows that microchannels only allow neurites to grow and pass, and induce directional growth of neurites. .
  • Figure 3 shows the trajectory tracking of glioma cells co-cultured with gliomas and hippocampal neurons under different drug treatments at different time points.
  • 3a shows the glioma cells without any drug treatment followed by Migration trajectory of neurites (control group).
  • Fig. 3b shows the migration trajectory of glioma cells following neurites after treatment with NGF-siRNA (experimental group).
  • Fig. 3c shows the glioma cells treated with BDNF-siRNA. Migration of stromal tumor cells with neurites (experimental group).
  • Figure 4 shows that the number of cancer cells in the same field of view of glioma cells treated with different drugs (blank, NGF-siRNA, BDNF-siRNA) is different, reflecting the effect of drug treatment (NGF-siRNA, BDNF-siRNA). Glioma cell migration ability was significantly reduced.
  • FIG. 5 shows the tracking of changes in the ability of panc-1 cells treated with different siRNAs along neurites in Test Example 2.
  • Fig. 6 shows the communication mechanism between different organelle networks and the corresponding image analysis algorithm.
  • Fig. 6a shows the cell recognition through vesicles
  • Fig. 6b shows the cell recognition through membrane fusion
  • Fig. 6c Cell recognition by membrane receptors is shown.
  • PDMS prepolymer purchased from Dow Corning, USA.
  • Dow Corning SYLGARD 184 silicone rubber is a two-component kit product consisting of liquid components, including basic components and curing agent. The basic components and curing agent are completely mixed at a weight ratio of 10: 1.
  • the glioma T98G cell line was purchased from Gaining Bio.
  • Pancreatic cancer panc-1 cell line and prostate cancer PC-3 cell line were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.
  • the colon cancer Caco-2 cell line was purchased from Kangwei Century, and the breast cancer MCF-7 cell line was purchased from KGI.
  • Hippocampal neuron and dorsal root ganglion (DRG) nerves were obtained from SD rats within 24 hours of birth (animals were provided by Viton Liwa).
  • DMEM medium DMEM / F12 medium, fetal bovine serum, monoclonal antibodies and pancreatin were purchased from Baoruyi (Beijing) Biotechnology Co., Ltd. under the brand name Invitrogen (Life Technologies).
  • Collagenase was purchased from Beijing Hongyue Innovation Technology Co., Ltd. under the brand name InVitro Scientific.
  • the DNase was purchased from Beijing Puyihua Technology Co., Ltd. under the brand Sigma.
  • Neurobasalmedium and N27 were purchased from Invitrogen (Shanghai) Trading Co., Ltd. under the brand name Invitrogen (Life Technologies).
  • This example is used to explain the preparation method of the drug screening platform of the present invention.
  • the microfluidic chip with high throughput mentioned in the present invention is shown in FIG. 1.
  • the size of this high-throughput microfluidic chip is specifically: the middle DRG nerve cell chamber is 4mm long, 4mm wide, and 100 ⁇ m thick; the size of the four cancer cell chambers is: 4mm long, 2mm wide, and 100 ⁇ m thick; cancer
  • the cell compartment is connected to the DRG nerve cell compartment through a 200 ⁇ m microchannel.
  • the width of the microchannel is 10 ⁇ m and the thickness is 10 ⁇ m.
  • the size of this microfluidic chip conforms to the 12-hole glass bottom plate of Invitro Scientific.
  • Auto CAD software was used to design the microfluidic chip according to the above dimensions, a mask was obtained by high-resolution printing, a template was prepared using photolithography, and a PDMS prepolymer (PDMS: crosslinking agent mass ratio 10: 1) was poured on the template. ) And placed in an oven at 80 ° C. to perform cross-linking polymerization, and the polymerized PDMS was peeled from the mask to form a PDMS-based microfluidic chip.
  • the microfluidic channels were formed on the two sides of each cell of the chip and sterilized in an autoclave (121 ° C, 15psi, 15min).
  • the cell culture conditions in the present invention are as follows:
  • the present invention uses glioma T98G cell line, pancreatic cancer panc-1 cell line, prostate cancer PC-3 cell line, colon cancer Caco-2 cell line, breast cancer MCF-7 cell line, hippocampal neurons and dorsal root nerve Ganglion (DRG) nerves have performed drug screening studies for neural-tumor cell recognition.
  • the culture environment of glioma T98G cells, pancreatic cancer panc-1 cells and breast cancer MCF-7 cells is: DMEM medium + 10% fetal bovine serum + 1% double antibody.
  • the culture environment of colon cancer Caco-2 cells is: DMEM medium + 20% fetal bovine serum + 1% double antibody.
  • the culture environment of prostate cancer PC-3 cell line was: DMEM / F12 medium + 10% fetal bovine serum + 1% double antibody.
  • Isolation and culture of hippocampal neurons SD rats were decapitated within 24 hours of birth on ice and the scalp, cartilage and meninges were cut open with arrow tweezers and separated to the sides to expose the brain tissue. The whole brain was tweezed Pick into a petri dish containing pre-chilled dissection fluid. Under the dissection microscope, open the two cerebral hemispheres with forceps, remove the capillaries around the hippocampus, carefully peel the hippocampus out and place it in another petri dish.
  • the hippocampus Separate the hippocampus into small 1 ⁇ 1mm pieces with tweezers, transfer to a centrifuge tube, digest with 0.25% trypsin in a 37 ° C water bath for 15 minutes, and shake the centrifuge tube every 5 minutes to fully contact the hippocampal tissue with the digestive fluid. . After the digestion is completed, the digestion is terminated with DMEM medium and gently rinsed twice. Pipette hippocampal tissue 5-10 times with a sterile pipette. Centrifuge the cell suspension (1300 rpm x 3min) and discard the supernatant. Resuspend the hippocampal neurons in the neuron culture medium (Neurobasalmedium + 10% N27 + 1% monoclonal antibody).
  • DRG neurons Isolation and culture of DRG neurons: SD rats were sacrificed and their spines removed within 24 hours of birth on ice. The spinal cord was exposed using sterile rongeurs, and the dorsal root ganglia distributed on both sides of the spinal cord were removed. , Put into pre-chilled DMEM medium. DRG tissue was gently rinsed with DMEM medium and transferred to a 15 mL centrifuge tube, digested with 10 ⁇ digestion solution (formula: trypsin 4mg / ml, collagenase 10mg / ml and DNase 1mg / ml) at 37 ° C for 30min, And shake the centrifuge tube every 5min to make the DRG tissue fully contact the digestive juice.
  • 10 ⁇ digestion solution formula: trypsin 4mg / ml, collagenase 10mg / ml and DNase 1mg / ml
  • DRG tissue was pipetted 5-10 times with a sterile pipette, the cell suspension was centrifuged (1300 rpm ⁇ 3min), and the supernatant was discarded.
  • the DRG neurons at the bottom were resuspended in neuronal medium (Neurobasalmedium + 10% N27 + 1% monoclonal antibody).
  • the drug pretreatment scheme for cancer cells is as follows:
  • siRNAs Different siRNAs (NGF-siRNA, BDNF-siRNA, GAL-siRNA, NT3-siRNA, nsRNA) were incubated with Lipofectamine 3000 at room temperature for 5 min (final siRNA concentration was 100 nM) to form liposome-siRNA complexes.
  • the above five different liposome-siRNA complexes were respectively incubated in pancreatic cancer panc-1 cells in a cell incubator for 48 h.
  • prostate cancer PC-3 cells, colon cancer Caco-2 cells, and breast cancer MCF-7 cells were treated in the same manner as described above.
  • the 12-well glass bottom plate of Invitro Scientific was treated with 100 ⁇ g / mL polylysine overnight, washed with PBS and dried, and the sterilized microfluidic chip was placed in a 12-well plate, so that the chip and the The bottom of the glass plate forms a sealed channel.
  • the neurons were injected into the DRG neuron cell of the chip at a density of 1 ⁇ 10 7 cells / mL, and cultured in a neuron medium for about 4 days. It was observed that the neurites can grow along 200 ⁇ m microchannels and reach cancer One side of the cell compartment.
  • add the drug-treated cancer cells in step 4 to different cancer cell compartments at a density of 6 ⁇ 10 6 cells / mL. After attaching them, use forceps Gently remove the chip and add neuronal medium to culture.
  • the 12-well plate with the chip removed was placed in a high-content imaging system, and the environmental control was set to 37 ° C and 5% CO 2 ; the cells were imaged continuously for 8 h using digital phase contrast mode.
  • Post-process the collected image trajectories perform statistical data analysis and data mining, establish a mathematical model of cell-cell mutual recognition, use biological image information technology to quantitatively characterize the protrusion and retraction of cancer cell edges, The migration of the tumor was quantitatively evaluated, and the morphological and dynamic change curve of the cancer cells was obtained.
  • This test example is used to illustrate the precise control of neurite growth by the microfluidic chip of the present invention.
  • the microfluidic chip in the present invention can realize precise control of neurite outgrowth, and the design of different chambers can realize co-culture of nerves and cancer cells. The results are shown in Figure 2.
  • this microfluidic chip has high-throughput characteristics, and it matches Invitro Scientific's 12-well plate, which can screen 48 different drugs at one time.
  • This test example is used to illustrate the screening of drugs by the microfluidic chip of the present invention.
  • Figure 6 shows the communication mechanism between different organelles. Each communication mechanism is set as an image analysis algorithm, and this algorithm can be used to analyze the cell recognition mechanism during the migration of cancer cells along the neurite.

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Abstract

Provided in the present invention are a high-throughput microfluidic chip, and a preparation method and the use thereof. The microfluidic chip comprises: a nerve cell chamber located in the center and cancer cell chambers located around the nerve cell chamber, and the nerve cell chamber and the cancer cell chambers are connected therebetween by microchannels. The microfluidic chip in the present invention can realize the precise control of neurite growth, and the design of different chambers can realize the co-culturing of nerves and cancer cells. In addition, this microfluidic chip has the characteristics of a high throughput, and the unique image analysis algorithm used by the present invention can evaluate the morphology of mutual recognition between the cancer cell and the neurite during the migration process of cancer cells.

Description

高通量微流控芯片、其制备方法及应用High-throughput microfluidic chip, preparation method and application thereof
相关申请的交叉引用Cross-reference to related applications
本申请要求2018年05月23日提交的第CN201811036755.1号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。This application claims priority from Chinese Invention Patent Application No. CN201811036755.1 filed on May 23, 2018, which is incorporated herein by reference in its entirety.
技术领域Technical field
本发明属于药物化学领域，具体涉及一种高通量微流控芯片，及其制备方法和应用。The invention belongs to the field of medicinal chemistry, and particularly relates to a high-throughput microfluidic chip, and a preparation method and application thereof.
背景技术Background technique
高通量的药物筛选是指在分子或细胞水平上，利用微板等作为筛选工具，以快速灵敏的自动化检测手段对大量的样品进行检测，因此对于新药的发现与评估具有重要意义。目前高通量的药物筛选主要借助微孔板实现，尽管其解决了传统药物筛选方法的耗时长、成本高等弊端，但仍然存在一些亟待解决的问题：基于微孔板的药物筛选由于细胞培养平台过于简单，不能模拟动物乃至人体内的细胞-细胞、细胞-微环境之间的相互作用。High-throughput drug screening refers to the use of microplates as a screening tool at the molecular or cell level to detect a large number of samples with rapid and sensitive automated detection methods. Therefore, it is of great significance for the discovery and evaluation of new drugs. At present, high-throughput drug screening is mainly achieved through microplates. Although it solves the disadvantages of long time and high cost of traditional drug screening methods, there are still some problems that need to be solved urgently: Drug screening based on microplates is due to the cell culture platform. Too simple to simulate cell-cell and cell-microenvironment interactions in animals and even humans.
与微孔板相比，微流控芯片操作所需的细胞量少，因其具有微米尺度空间以及相对独立、封闭环境等优点，在药物筛选等领域应用广泛。目前，微流控芯片细胞水平的药物筛选主要通过以下几种方式：(1)基于灌流模式的高通量筛选。(2)基于微液滴的高通量筛选。(3)微阵列模式的高通量筛选。虽然以上技术手段能够提高药物筛选的通量，但是对于研究细胞-细胞，细胞-微环境的相互作用方面来说，这些微流控芯片的设计仍然不能实现对细胞微环境进行精确控制和操纵。另外，对于细胞间识别成像分析来说，现有的基于微流控芯片的药物筛选主要通过荧光强弱等信号读出，且图像分析参数单一，速度低。因此，目前的基于微流控细胞水平的药物筛选平台在细胞-微环境的相互识别及图像分析方面仍然存在很大的局限性。Compared with microplates, the microfluidic chip requires fewer cells to operate. Because of its micrometer-scale space and relatively independent, closed environment, it is widely used in drug screening and other fields. At present, the drug screening at the microfluidic chip level is mainly through the following methods: (1) High-throughput screening based on perfusion mode. (2) High-throughput screening based on microdroplets. (3) High-throughput screening in microarray mode. Although the above technical methods can improve the throughput of drug screening, for the study of cell-cell and cell-microenvironment interactions, the design of these microfluidic chips still cannot achieve precise control and manipulation of the cell microenvironment. In addition, for inter-cell recognition imaging analysis, the existing microfluidic chip-based drug screening mainly reads out signals such as fluorescence intensity, and the image analysis parameters are single and low speed. Therefore, current drug screening platforms based on microfluidic cells still have significant limitations in cell-microenvironment mutual recognition and image analysis.
先前的文献报道了一种可以进行用于神经元及胶质细胞共培养的微流控芯片，用于研究胶质细胞对神经细胞转染效率的影响。尽管这种微流控芯片能够实现不同细胞的共培养，但是不能够满足药物筛选的高通量需求。此外，传统的基于微流控芯片的药物筛选，在表征药物代谢及细胞毒性的过程中，通常使用荧光信号进行表征，或者将芯片与ESI-Q-TOF质谱分析仪联用。虽然这种微流控芯片与质谱联用的筛选平台能够实现药物筛选的目的，但是采用质谱进行药物代谢的评价具有很大的局限性，一方面，质谱分析手段繁琐，尤其对于蛋白质的分析，质谱分析过程繁琐，适用范围窄。另一方面，现有方法图像分析参数单一，速度低且难以定量。Previous literature reported a microfluidic chip that can be used for co-culture of neurons and glial cells to study the effect of glial cells on the transfection efficiency of neurons. Although this microfluidic chip can achieve co-culture of different cells, it cannot meet the high-throughput needs of drug screening. In addition, traditional microfluidic chip-based drug screening usually uses fluorescence signals for characterization of drug metabolism and cytotoxicity, or the chip is used in combination with an ESI-Q-TOF mass spectrometer. Although this kind of microfluidic chip and mass spectrometry screening platform can achieve the purpose of drug screening, the use of mass spectrometry for drug metabolism evaluation has great limitations. On the one hand, mass spectrometry analysis methods are complicated, especially for protein analysis. Mass spectrometric analysis is cumbersome and has a narrow application range. On the other hand, the existing methods have single image analysis parameters, low speed and difficult to quantify.
发明内容Summary of the Invention
因此，本发明的目的在于克服现有技术中的缺陷，提供一种高通量微流控芯片，及其制备方法和应用。Therefore, the purpose of the present invention is to overcome the defects in the prior art, and provide a high-throughput microfluidic chip, and a preparation method and application thereof.
在阐述本发明内容之前，定义本文中所使用的术语如下：Before describing the present invention, the terms used herein are defined as follows:
术语“PDMS”是指：聚二甲基硅氧烷。The term "PDMS" means: polydimethylsiloxane.
为实现上述目的，本发明的第一方面提供了一种高通量微流控芯片，所述微流控芯片包括：位于中央的神经细胞小室和位于所述神经细胞小室周围的癌细胞小室，所述神经细胞小室和癌细胞小室中间通过微通道连接；In order to achieve the above object, a first aspect of the present invention provides a high-throughput microfluidic chip, the microfluidic chip includes a central nerve cell chamber and a cancer cell chamber surrounding the neural cell chamber, The middle of the nerve cell compartment and the cancer cell compartment is connected by a microchannel;
优选地，所述微流控芯片包括2～6个癌细胞小室，最优选为4个。Preferably, the microfluidic chip includes two to six cancer cell compartments, and most preferably four.
根据本发明第一方面的微流控芯片，其中，所述神经细胞小室的长度为1～10mm，优选为2～6mm，最优选为4mm；The microfluidic chip according to the first aspect of the present invention, wherein the length of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
所述神经细胞小室的宽度为1～10mm，优选为2～6mm，最优选为4mm；和/或The width of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm; and / or
所述神经细胞小室的厚度为10～200μm，优选为50～150μm，最优选为100μm。The thickness of the nerve cell compartment is 10 to 200 μm, preferably 50 to 150 μm, and most preferably 100 μm.
根据本发明第一方面的微流控芯片，其中，所述癌细胞小室的长度为1～10mm，优选为2～6mm，最优选为4mm；The microfluidic chip according to the first aspect of the present invention, wherein the length of the cancer cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
所述癌细胞小室的宽度为1～10mm，优选为1～3mm，最优选为2mm；和/或The width of the cancer cell compartment is 1 to 10 mm, preferably 1 to 3 mm, and most preferably 2 mm; and / or
所述癌细胞小室的厚度为10～200μm，优选为50～150μm，最优选为100μm。The thickness of the cancer cell compartment is 10 to 200 μm, preferably 50 to 150 μm, and most preferably 100 μm.
根据本发明第一方面的微流控芯片，其中，所述微通道的长度为100～500μm，优选为100～300μm，最优选为200μm；The microfluidic chip according to the first aspect of the present invention, wherein the length of the microchannel is 100 to 500 μm, preferably 100 to 300 μm, and most preferably 200 μm;
所述微通道的宽度为5～50μm，优选为5～20μm，最优选为10μm；和/或The width of the microchannel is 5-50 μm, preferably 5-20 μm, and most preferably 10 μm; and / or
所述微通道的厚度为5～50μm，优选为5～20μm，最优选为10μm。The thickness of the microchannel is 5 to 50 μm, preferably 5 to 20 μm, and most preferably 10 μm.
本发明的第二方面提供了第一方面所述的微流控芯片的制备方法，所述方法包括以下步骤：A second aspect of the present invention provides a method for preparing a microfluidic chip according to the first aspect, the method including the following steps:
(1)设计微流控芯片，打印掩膜版；(1) Design microfluidic chip and print mask version;
(2)利用光刻技术制备模板；(2) using a photolithography technique to prepare a template;
(3)在模板上浇筑PDMS预聚物，加热，从掩膜版上剥下所述PDMS得到所述微流控芯片。(3) Pouring PDMS prepolymer on the template, heating, and peeling the PDMS from the mask to obtain the microfluidic chip.
根据本发明第二方面的制备方法，其中，所述步骤(3)中，所述PDMS预聚物中，PDMS与交联剂的质量比为5～20:1，优选为5～15:1，最优选为10:1。The preparation method according to the second aspect of the present invention, wherein, in the step (3), the mass ratio of PDMS to the cross-linking agent in the PDMS prepolymer is 5 to 20: 1, preferably 5 to 15: 1 , Most preferably 10: 1.
根据本发明第二方面的制备方法，其中，所述步骤(3)中，所述加热温度为50～150℃，优选为50～100℃，最优选为80℃。The preparation method according to the second aspect of the present invention, wherein in the step (3), the heating temperature is 50 to 150 ° C, preferably 50 to 100 ° C, and most preferably 80 ° C.
本发明的第三方面提供了一种高通量药物筛选平台，所述平台包括：第一方面所述的微流控芯片和高内涵成像系统。A third aspect of the present invention provides a high-throughput drug screening platform, which includes the microfluidic chip and high-content imaging system described in the first aspect.
本发明的第四方面提供了第一方面所述的第一方面所述的微流控芯片或第三方面所述的高通量药物筛选平台在制备药物筛选产品尤其是高通量药物筛选产品中的应用。The fourth aspect of the present invention provides the microfluidic chip according to the first aspect or the high-throughput drug screening platform according to the third aspect, in preparing a drug screening product, especially a high-throughput drug screening product. Application.
根据本发明第四方面的应用，其中，所述药物为神经-肿瘤微环境相关药物。According to the application of the fourth aspect of the present invention, the drug is a neuro-tumor microenvironment-related drug.
本发明的第五方面提供了一种药物筛选方法，所述方法采用：A fifth aspect of the present invention provides a method for screening a drug, the method using:
第一方面所述的微流控芯片；和/或The microfluidic chip according to the first aspect; and / or
第三方面所述的高通量药物筛选平台。The high-throughput drug screening platform described in the third aspect.
本发明的第六方面提供了一种细胞共培养装置，所述装置包括第一方面所述的微流控芯片。A sixth aspect of the present invention provides a cell co-culture device, the device comprising the microfluidic chip described in the first aspect.
本发明的第七方面提供了一种细胞共培养方法，所述方法采用第一方面所述的微流控芯片。A seventh aspect of the present invention provides a cell co-culture method, which uses the microfluidic chip described in the first aspect.
本发明的第八方面提供了一种细胞识别平台，所述平台包括第一方面所述的微流控芯片；An eighth aspect of the present invention provides a cell recognition platform including the microfluidic chip described in the first aspect;
优选地，所述细胞识别为细胞-细胞识别和/或细胞-微环境识别。Preferably, the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
本发明的第九方面提供了一种细胞识别方法，所述方法采用第一方面所述的微流控芯片；A ninth aspect of the present invention provides a cell identification method, which uses the microfluidic chip described in the first aspect;
优选地，所述细胞识别为细胞-细胞识别和/或细胞-微环境识别。Preferably, the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
本发明的目的是基于一种具有高通量特性的微流控芯片，作为细胞-细胞、细胞-微环境识别的平台，利用高内涵成像系统及一种特殊的计算机图像分析算法对癌细胞与神经突之间的相互作用进行批量分析统计，以期成功筛选出与神经-肿瘤微环境相关的抗癌药物。The object of the present invention is based on a microfluidic chip with high-throughput characteristics. As a platform for cell-to-cell and cell-to-microenvironment recognition, a high-content imaging system and a special computer image analysis algorithm are used to detect cancer cells and cells. The interactions between neurites were analyzed in batches in order to successfully screen anticancer drugs related to the neuro-tumor microenvironment.
根据文献报道，许多癌细胞分泌神经生长因子(如NGF等)及神经营养因子(如BDNF，GAL，NT-3等)，这些细胞因子的表达与神经——肿瘤微环境的形成密切相关。本发明基于一种具有微通道的微流控芯片，作为癌细胞——神经突相互识别的平台，并 结合高内涵成像分析系统以及独特的图像分析算法，对不同的siRNA进行高通量筛选。According to literature reports, many cancer cells secrete nerve growth factors (such as NGF, etc.) and neurotrophic factors (such as BDNF, GAL, NT-3, etc.). The expression of these cytokines is closely related to the formation of the neuro-tumor microenvironment. The invention is based on a microfluidic chip with microchannels, as a platform for mutual recognition of cancer cells and neurites, and combined with a high-content imaging analysis system and a unique image analysis algorithm, high-throughput screening of different siRNAs.
本发明中的微流控芯片不仅具有可以提供神经突定向生长的微通道，可以实现不同细胞的共培养。而且这种芯片具有五个小室，在中心小室培养神经元，四周小室进行不同药物筛选，并且结合12孔板，可以一次性实现48种不同的药物，具有高通量的特点。The microfluidic chip in the present invention not only has microchannels that can provide directional growth of neurites, but also can realize co-culture of different cells. In addition, this chip has five chambers. Neurons are cultured in the central chamber, and different chambers are screened for different drugs. Combined with a 12-well plate, 48 different drugs can be realized at one time, which has the characteristics of high throughput.
本发明中的微流控芯片可以实现对神经突生长的精确控制，不同小室的设计可以实现对神经与癌细胞的共培养。此外，这种微流控芯片具有高通量的特点，与In Vitro Scientific公司的12孔板相匹配，可以一次性筛选48种不同的药物。此外，本发明采用的独特图像分析算法，可以对癌细胞迁移过程中与神经突间相互识别的形态进行评价。The microfluidic chip in the present invention can realize precise control of neurite outgrowth, and the design of different chambers can realize co-culture of nerves and cancer cells. In addition, this microfluidic chip has high-throughput characteristics, and it matches Invitro Scientific's 12-well plate, which can screen 48 different drugs at one time. In addition, the unique image analysis algorithm used in the present invention can evaluate the morphology of mutual recognition between neurites during cancer cell migration.
本发明中的微流控芯片作为一个细胞共培养的高通量分析平台，结合高内涵细胞，建立了体外高通量药物筛选平台，对于细胞进行自动的实时成像观察，实现了操作的便捷性；此外，本发明采用独特的图像分析算法，可满足药物研发的所有需求。The microfluidic chip of the present invention is used as a high-throughput analysis platform for cell co-culture, and combined with high-content cells, an in vitro high-throughput drug screening platform is established, and automatic real-time imaging observation of the cells is realized to realize the convenience of operation In addition, the present invention uses a unique image analysis algorithm to meet all the needs of drug development.
本发明的高通量微流控芯片可以具有但不限于以下有益效果：The high-throughput microfluidic chip of the present invention can have, but is not limited to, the following beneficial effects:
本发明涉及采用微流控芯片联合高内涵成像系统进行高通量药物筛选的领域，可以用于筛选与神经-肿瘤微环境相关的抗肿瘤药物(包括小分子抗癌药物、siRNA、CRISPR/Cas9体系等)，并且可以对同一药物在不同类别癌细胞体系中的抗肿瘤效果进行评价，这种基于高通量的微流控芯片的细胞间识别成像的筛选体系使得药物筛选的速度大大提高，节约了时间和成本。本发明在高通量药物筛选领域有广阔的应用前景。此外，本发明中所应用的图像分析算法对于研究肿瘤微环境中细胞间相互作用的机理具有重要意义。The invention relates to the field of high-throughput drug screening using a microfluidic chip combined with a high-content imaging system, and can be used for screening antitumor drugs (including small molecule anticancer drugs, siRNA, CRISPR / Cas9) related to the neuro-tumor microenvironment. System, etc.), and can evaluate the anti-tumor effect of the same drug in different types of cancer cell systems. This high-throughput microfluidic chip-based screening system for inter-cell recognition imaging makes the speed of drug screening greatly improved. Saves time and costs. The invention has broad application prospects in the field of high-throughput drug screening. In addition, the image analysis algorithm applied in the present invention is of great significance for studying the mechanism of cell-cell interaction in the tumor microenvironment.
附图的简要说明Brief description of the drawings
以下，结合附图来详细说明本发明的实施方案，其中：Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, in which:
图1示出了本发明高通量的微流控芯片示意图。FIG. 1 shows a schematic diagram of a high-throughput microfluidic chip of the present invention.
图2示出了试验例1中本发明高通量的微流控芯片不同小室对神经与癌细胞的共培养，其中图2a示出了本发明中高通量的微流控芯片实物图,该芯片的尺寸恰好可以放入12孔板中，图2b示出了海马神经元与胶质瘤细胞的共培养，图2c示出了微通道只允许神经突生长和通过，诱导神经突的定向生长。FIG. 2 shows the co-culture of nerves and cancer cells in different chambers of the high-throughput microfluidic chip of the present invention in Experimental Example 1. FIG. 2a shows a physical picture of the high-throughput microfluidic chip of the present invention. The size of the chip can just fit into a 12-well plate. Figure 2b shows co-culture of hippocampal neurons and glioma cells, and Figure 2c shows that microchannels only allow neurites to grow and pass, and induce directional growth of neurites. .
图3示出了不同药物处理下的胶质瘤与海马神经元共培养，不同时间点下胶质瘤细胞的迁移轨迹的追踪，其中3a示出了未经任何药物处理的胶质瘤细胞随神经突的迁移轨迹(对照组)，图3b示出了经过NGF-siRNA处理后的胶质瘤细胞随神经突的迁移轨迹(实验组)，图3c示出了经过BDNF-siRNA处理后的胶质瘤细胞随神经突的迁移轨迹(实验组)。Figure 3 shows the trajectory tracking of glioma cells co-cultured with gliomas and hippocampal neurons under different drug treatments at different time points. 3a shows the glioma cells without any drug treatment followed by Migration trajectory of neurites (control group). Fig. 3b shows the migration trajectory of glioma cells following neurites after treatment with NGF-siRNA (experimental group). Fig. 3c shows the glioma cells treated with BDNF-siRNA. Migration of stromal tumor cells with neurites (experimental group).
图4示出了不同药物处理(空白，NGF-siRNA，BDNF-siRNA)的胶质瘤细胞，在相同视野大小内的癌细胞数量不同，反映了药物处理(NGF-siRNA，BDNF-siRNA)后 胶质瘤细胞迁移能力明显减弱。Figure 4 shows that the number of cancer cells in the same field of view of glioma cells treated with different drugs (blank, NGF-siRNA, BDNF-siRNA) is different, reflecting the effect of drug treatment (NGF-siRNA, BDNF-siRNA). Glioma cell migration ability was significantly reduced.
图5示出了试验例2中不同siRNA处理过的panc-1细胞沿神经突迁移能力变化的追踪。FIG. 5 shows the tracking of changes in the ability of panc-1 cells treated with different siRNAs along neurites in Test Example 2. FIG.
图6示出了不同细胞器网络之间的通讯机制以及与之对应的图像分析算法，其中图6a示出了通过囊泡进行的细胞识别，图6b示出了通过膜融合的细胞识别，图6c示出了通过膜受体的细胞识别。Fig. 6 shows the communication mechanism between different organelle networks and the corresponding image analysis algorithm. Fig. 6a shows the cell recognition through vesicles, Fig. 6b shows the cell recognition through membrane fusion, and Fig. 6c Cell recognition by membrane receptors is shown.
实施发明的最佳方式The best way to implement the invention
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。The present invention will be further described below through specific examples, but it should be understood that these examples are only used for more detailed and specific explanation, and should not be understood to limit the present invention in any form.
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。This section provides a general description of the materials and test methods used in the tests of the present invention. Although many materials and methods of operation used to achieve the purpose of the present invention are well known in the art, the present invention is still described here in as much detail as possible. It is clear to a person skilled in the art that in this context, unless otherwise specified, the materials and methods of operation of the present invention are well known in the art.
以下实施例中使用的试剂和仪器如下：The reagents and instruments used in the following examples are as follows:
试剂：Reagent:
PDMS预聚物，购自美国道康宁，道康宁SYLGARD 184硅橡胶由液体组分组成的双组分套件产品，包括基本组分与固化剂，基本组分与固化剂按10:1重量比完全混合。PDMS prepolymer, purchased from Dow Corning, USA. Dow Corning SYLGARD 184 silicone rubber is a two-component kit product consisting of liquid components, including basic components and curing agent. The basic components and curing agent are completely mixed at a weight ratio of 10: 1.
胶质瘤T98G细胞系购自盖宁生物，The glioma T98G cell line was purchased from Gaining Bio.
胰腺癌panc-1细胞系及前列腺癌PC-3细胞系均购自北京鼎国昌盛生物技术有限公司Pancreatic cancer panc-1 cell line and prostate cancer PC-3 cell line were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.
结肠癌Caco-2细胞系购自康为世纪，乳腺癌MCF-7细胞系购自凯基生物。The colon cancer Caco-2 cell line was purchased from Kangwei Century, and the breast cancer MCF-7 cell line was purchased from KGI.
海马神经元神经及背根神经节(DRG)神经取自出生24小时内的SD大鼠(动物由维通利华提供)。Hippocampal neuron and dorsal root ganglion (DRG) nerves were obtained from SD rats within 24 hours of birth (animals were provided by Viton Liwa).
DMEM培养基，DMEM/F12培养基，胎牛血清，双抗及胰酶均购自宝如亿(北京)生物技术有限公司，品牌为Invitrogen(Life Technologies)。DMEM medium, DMEM / F12 medium, fetal bovine serum, monoclonal antibodies and pancreatin were purchased from Baoruyi (Beijing) Biotechnology Co., Ltd. under the brand name Invitrogen (Life Technologies).
胶原酶购自北京鸿跃创新科技有限公司，品牌为In Vitro Scientific。Collagenase was purchased from Beijing Hongyue Innovation Technology Co., Ltd. under the brand name InVitro Scientific.
DNA酶购自北京普益华科技有限公司，品牌为Sigma。The DNase was purchased from Beijing Puyihua Technology Co., Ltd. under the brand Sigma.
Neurobasal medium及N27均购自英潍捷基(上海)贸易有限公司，品牌为Invitrogen(Life Technologies)。Both Neurobasalmedium and N27 were purchased from Invitrogen (Shanghai) Trading Co., Ltd. under the brand name Invitrogen (Life Technologies).
实施例1Example 1
本实施例用于说明本发明药物筛选平台的制备方法。This example is used to explain the preparation method of the drug screening platform of the present invention.
1、微流控芯片的结构。1. Structure of microfluidic chip.
本发明中提及的具有高通量的微流控芯片如图1所示。The microfluidic chip with high throughput mentioned in the present invention is shown in FIG. 1.
这种高通量的微流控芯片尺寸具体为：中间DRG神经细胞小室长为4mm，宽为4mm，厚度为100μm；四个癌细胞小室的尺寸为：长4mm，宽2mm，厚度100μm；癌细胞小室与DRG神经细胞小室中间通过200μm的微通道连接，微通道的宽为10μm，厚度为10μm。这种微流控芯片尺寸大小符合In Vitro Scientific公司的12孔玻璃底板。The size of this high-throughput microfluidic chip is specifically: the middle DRG nerve cell chamber is 4mm long, 4mm wide, and 100 μm thick; the size of the four cancer cell chambers is: 4mm long, 2mm wide, and 100 μm thick; cancer The cell compartment is connected to the DRG nerve cell compartment through a 200 μm microchannel. The width of the microchannel is 10 μm and the thickness is 10 μm. The size of this microfluidic chip conforms to the 12-hole glass bottom plate of Invitro Scientific.
2、高通量的微流控芯片的具体制作步骤如下：2. The specific manufacturing steps of the high-throughput microfluidic chip are as follows:
采用auto CAD软件按照上述尺寸设计微流控芯片，采用高分辨率打印得到掩膜版，利用光刻技术制备模板，在模板上面浇筑PDMS预聚物(PDMS：交联剂质量比为10:1)并置于80℃烘箱使其交联聚合，将聚合后的PDMS从掩膜版上剥下，从而PDMS材质的微流控芯片。在芯片的每个小室两侧进行打孔形成微流控通道，并置于高压灭菌锅中灭菌(121℃，15psi，15min)。Auto CAD software was used to design the microfluidic chip according to the above dimensions, a mask was obtained by high-resolution printing, a template was prepared using photolithography, and a PDMS prepolymer (PDMS: crosslinking agent mass ratio 10: 1) was poured on the template. ) And placed in an oven at 80 ° C. to perform cross-linking polymerization, and the polymerized PDMS was peeled from the mask to form a PDMS-based microfluidic chip. The microfluidic channels were formed on the two sides of each cell of the chip and sterilized in an autoclave (121 ° C, 15psi, 15min).
3、本发明中细胞培养条件如下：3. The cell culture conditions in the present invention are as follows:
本发明采用了胶质瘤T98G细胞系，胰腺癌panc-1细胞系，前列腺癌PC-3细胞系，结肠癌Caco-2细胞系，乳腺癌MCF-7细胞系，海马神经元以及背根神经节(DRG)神经进行了神经-肿瘤细胞间识别的药物筛选研究。The present invention uses glioma T98G cell line, pancreatic cancer panc-1 cell line, prostate cancer PC-3 cell line, colon cancer Caco-2 cell line, breast cancer MCF-7 cell line, hippocampal neurons and dorsal root nerve Ganglion (DRG) nerves have performed drug screening studies for neural-tumor cell recognition.
胶质瘤T98G细胞，胰腺癌panc-1细胞及乳腺癌MCF-7细胞的培养环境为：DMEM培养基+10％胎牛血清+1％双抗。The culture environment of glioma T98G cells, pancreatic cancer panc-1 cells and breast cancer MCF-7 cells is: DMEM medium + 10% fetal bovine serum + 1% double antibody.
结肠癌Caco-2细胞的培养环境为：DMEM培养基+20％胎牛血清+1％双抗。The culture environment of colon cancer Caco-2 cells is: DMEM medium + 20% fetal bovine serum + 1% double antibody.
前列腺癌PC-3细胞系的培养环境为：DMEM/F12培养基+10％胎牛血清+1％双抗。The culture environment of prostate cancer PC-3 cell line was: DMEM / F12 medium + 10% fetal bovine serum + 1% double antibody.
海马神经元的分离与培养：于冰上将出生24h内的SD系乳鼠断头处死并用箭头镊将头皮、软骨和脑膜剪开并向两侧分离，使脑组织暴露，用镊子将整个大脑挑至盛有预冷解剖液的培养皿中。在解剖镜下用镊子将两个大脑半球翻开，夹除海马区周围的毛细血管，将海马小心剥离出来放至另一培养皿中。用镊子将海马分离成1×1mm的小块，转至离心管中，用0.25％的胰酶于37℃水浴锅中消化15min，并每隔5min摇晃一次离心管使海马组织与消化液充分接触。消化结束后，用DMEM培养基终止消化并轻轻漂洗两遍。利用无菌吸管对海马组织进行吹打5-10次，将细胞悬液离心(1300rpm×3min)后弃上清，底部的海马神经元重悬于神经元培养基中(Neurobasal medium+10％N27+1％双抗)。Isolation and culture of hippocampal neurons: SD rats were decapitated within 24 hours of birth on ice and the scalp, cartilage and meninges were cut open with arrow tweezers and separated to the sides to expose the brain tissue. The whole brain was tweezed Pick into a petri dish containing pre-chilled dissection fluid. Under the dissection microscope, open the two cerebral hemispheres with forceps, remove the capillaries around the hippocampus, carefully peel the hippocampus out and place it in another petri dish. Separate the hippocampus into small 1 × 1mm pieces with tweezers, transfer to a centrifuge tube, digest with 0.25% trypsin in a 37 ° C water bath for 15 minutes, and shake the centrifuge tube every 5 minutes to fully contact the hippocampal tissue with the digestive fluid. . After the digestion is completed, the digestion is terminated with DMEM medium and gently rinsed twice. Pipette hippocampal tissue 5-10 times with a sterile pipette. Centrifuge the cell suspension (1300 rpm x 3min) and discard the supernatant. Resuspend the hippocampal neurons in the neuron culture medium (Neurobasalmedium + 10% N27 + 1% monoclonal antibody).
DRG神经元的分离与培养：于冰上将出生24h内的SD系乳鼠断头处死并取其脊椎，利用无菌咬骨钳暴露脊髓，将分布于脊髓两侧的背根神经节取下，放入预冷的DMEM培养基中。用DMEM培养基轻轻漂洗DRG组织并转移至15mL离心管中，用10×消化液(配方为：胰酶4mg/ml，胶原酶10mg/ml及DNA酶1mg/ml)于37℃消化30min，并每隔5min摇晃一次离心管使DRG组织与消化液充分接触。消化结束后，用DMEM培 养基终止消化并轻轻漂洗三遍。利用无菌吸管对DRG组织进行吹打5-10次，将细胞悬液离心(1300rpm×3min)后弃上清，底部的DRG神经元重悬于神经元培养基中(Neurobasal medium+10％N27+1％双抗)。Isolation and culture of DRG neurons: SD rats were sacrificed and their spines removed within 24 hours of birth on ice. The spinal cord was exposed using sterile rongeurs, and the dorsal root ganglia distributed on both sides of the spinal cord were removed. , Put into pre-chilled DMEM medium. DRG tissue was gently rinsed with DMEM medium and transferred to a 15 mL centrifuge tube, digested with 10 × digestion solution (formula: trypsin 4mg / ml, collagenase 10mg / ml and DNase 1mg / ml) at 37 ° C for 30min, And shake the centrifuge tube every 5min to make the DRG tissue fully contact the digestive juice. After digestion is complete, stop digestion with DMEM medium and rinse gently three times. DRG tissue was pipetted 5-10 times with a sterile pipette, the cell suspension was centrifuged (1300 rpm × 3min), and the supernatant was discarded. The DRG neurons at the bottom were resuspended in neuronal medium (Neurobasalmedium + 10% N27 + 1% monoclonal antibody).
4.癌细胞的药物预处理方案如下：4. The drug pretreatment scheme for cancer cells is as follows:
将不同的siRNA(NGF-siRNA，BDNF-siRNA，GAL-siRNA，NT3-siRNA，nsRNA)分别与Lipofectamine 3000室温孵育5min(siRNA的终浓度为100nM)，使其形成脂质体-siRNA复合物。将上述五种不同的脂质体-siRNA复合物分别于胰腺癌panc-1细胞于细胞培养箱中孵育48h。此外，前列腺癌PC-3细胞，结肠癌Caco-2细胞，乳腺癌MCF-7细胞的药物处理方式同上所述。Different siRNAs (NGF-siRNA, BDNF-siRNA, GAL-siRNA, NT3-siRNA, nsRNA) were incubated with Lipofectamine 3000 at room temperature for 5 min (final siRNA concentration was 100 nM) to form liposome-siRNA complexes. The above five different liposome-siRNA complexes were respectively incubated in pancreatic cancer panc-1 cells in a cell incubator for 48 h. In addition, prostate cancer PC-3 cells, colon cancer Caco-2 cells, and breast cancer MCF-7 cells were treated in the same manner as described above.
5.微流控芯片联合高内涵系统的药物筛选平台的建立5. Establishment of drug screening platform with microfluidic chip combined with high content system
将In Vitro Scientific公司的12孔玻璃底板用100μg/mL的多聚赖氨酸进行过夜处理后用PBS洗涤并晾干，将灭菌后的微流控芯片置于12孔板中，使芯片与玻璃板底形成密封的通道。The 12-well glass bottom plate of Invitro Scientific was treated with 100 μg / mL polylysine overnight, washed with PBS and dried, and the sterilized microfluidic chip was placed in a 12-well plate, so that the chip and the The bottom of the glass plate forms a sealed channel.
将神经元以1×10 7cells/mL的密度注入到芯片的DRG神经细胞小室中，采用神经元培养基培养4天左右，可以观察到神经突能够沿着200μm的微通道进行生长并到达癌细胞小室的一侧。当神经突生长至癌细胞小室时，将第4步中药物预处理过的癌细胞以6×10 6cells/mL的密度分别加到不同的癌细胞小室中，待其贴壁后，用镊子轻轻将芯片移除，加入神经元培养基培养。将移除芯片的12孔板放至高内涵成像系统中，环境控制设置为37℃，5％CO 2；采用数字相差模式对细胞进行连续8h成像观察。 The neurons were injected into the DRG neuron cell of the chip at a density of 1 × 10 7 cells / mL, and cultured in a neuron medium for about 4 days. It was observed that the neurites can grow along 200 μm microchannels and reach cancer One side of the cell compartment. When the neurites grow into the cancer cell compartment, add the drug-treated cancer cells in step 4 to different cancer cell compartments at a density of 6 × 10 6 cells / mL. After attaching them, use forceps Gently remove the chip and add neuronal medium to culture. The 12-well plate with the chip removed was placed in a high-content imaging system, and the environmental control was set to 37 ° C and 5% CO 2 ; the cells were imaged continuously for 8 h using digital phase contrast mode.
6.神经-肿瘤细胞间识别的图像分析6. Image analysis of neural-tumor cell recognition
进入高内涵“harmony”软件的“Image Analysis”模块，选用“Cell Migration:Tracking”分析模块，对癌细胞8h内的迁移能力进行评估，可以得到不同药物对癌细胞迁移能力的影响。Enter the "Image Analysis" module of the high content "harmony" software and select the "Cell Migration: Tracking" analysis module to evaluate the migration ability of cancer cells within 8 hours. You can get the effect of different drugs on the cancer cell migration ability.
对采集到的图像轨迹进行后处理，进行统计数据分析及数据挖掘，建立细胞-细胞相互识别的数学模型，使用生物图像信息学技术定量表征癌细胞边缘的突出和回缩，对肿瘤细胞沿神经突的迁移进行定量评价，得出癌细胞的形态动力学变化曲线。Post-process the collected image trajectories, perform statistical data analysis and data mining, establish a mathematical model of cell-cell mutual recognition, use biological image information technology to quantitatively characterize the protrusion and retraction of cancer cell edges, The migration of the tumor was quantitatively evaluated, and the morphological and dynamic change curve of the cancer cells was obtained.
试验例1Test example 1
本试验例用于说明本发明微流控芯片对神经突生长的精确控制。This test example is used to illustrate the precise control of neurite growth by the microfluidic chip of the present invention.
本发明中的微流控芯片可以实现对神经突生长的精确控制，不同小室的设计可以实现对神经与癌细胞的共培养。结果如图2所示。此外，这种微流控芯片具有高通量的特点，与In Vitro Scientific公司的12孔板相匹配，可以一次性筛选48种不同的药物。The microfluidic chip in the present invention can realize precise control of neurite outgrowth, and the design of different chambers can realize co-culture of nerves and cancer cells. The results are shown in Figure 2. In addition, this microfluidic chip has high-throughput characteristics, and it matches Invitro Scientific's 12-well plate, which can screen 48 different drugs at one time.
试验例2Test example 2
本试验例用于说明本发明微流控芯片对药物的筛选。This test example is used to illustrate the screening of drugs by the microfluidic chip of the present invention.
通过这种微流控芯片进行胶质瘤-海马神经元共培养，并成功筛选出能够抑制肿瘤研神经突迁移的NGF-siRNA及BDNF-siRNA，结果如图3所示。此外，通过与高内涵系统联用，可以对药物筛选的结果进行定量评价，结果如图4所示。将微流控芯片与高内涵联用，增强了图像分析的速度和通量，可以对每个细胞进行轨迹追踪。图5为不同siRNA处理过的panc-1细胞沿神经突迁移能力变化的追踪，点越分散，表明细胞迁移能力越强。从图5可以看出，经NGF-siRNA处理过的panc-1细胞迁移能力明显减弱。Glioma- hippocampal neuron co-culture was performed using this microfluidic chip, and NGF-siRNA and BDNF-siRNA that can inhibit tumor research neurite migration were successfully selected. The results are shown in FIG. 3. In addition, by combining with the high-content system, the results of drug screening can be quantitatively evaluated. The results are shown in Figure 4. The combination of microfluidic chip and high content enhances the speed and throughput of image analysis, and can track the trajectory of each cell. Figure 5 is a trace of the changes in the ability of panc-1 cells treated with different siRNAs along the neurite. The more scattered the points, the stronger the cell migration ability. It can be seen from Fig. 5 that the migration ability of panc-1 cells treated with NGF-siRNA was significantly reduced.
通过使用图像分析算法，可以对癌细胞迁移过程中与神经突间相互识别的形态进行评价。图6示出了不同细胞器之间的通讯机制，将每一种通讯机制设置为一种图像分析算法，利用这种算法可以分析癌细胞沿神经突迁移过程中的细胞识别机制。By using image analysis algorithms, the morphology of mutual recognition between neurites during cancer cell migration can be evaluated. Figure 6 shows the communication mechanism between different organelles. Each communication mechanism is set as an image analysis algorithm, and this algorithm can be used to analyze the cell recognition mechanism during the migration of cancer cells along the neurite.
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。Although the present invention has been described to a certain degree, it is apparent that appropriate changes can be made in various conditions without departing from the spirit and scope of the invention. It will be understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which includes equivalent replacements of each of the factors mentioned.

Claims (15)

  1. 一种高通量微流控芯片，其特征在于，所述微流控芯片包括：位于中央的神经细胞小室和位于所述神经细胞小室周围的癌细胞小室，所述神经细胞小室和癌细胞小室中间通过微通道连接；A high-throughput microfluidic chip, characterized in that the microfluidic chip includes a nerve cell chamber located in the center and a cancer cell chamber located around the nerve cell chamber, the nerve cell chamber and a cancer cell chamber. The middle is connected through a micro channel;
    优选地，所述微流控芯片包括2～6个癌细胞小室，最优选为4个。Preferably, the microfluidic chip includes two to six cancer cell compartments, and most preferably four.
  2. 根据权利要求1所述的微流控芯片，其特征在于，所述神经细胞小室的长度为1～10mm，优选为2～6mm，最优选为4mm；The microfluidic chip according to claim 1, wherein the length of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
    所述神经细胞小室的宽度为1～10mm，优选为2～6mm，最优选为4mm；和/或The width of the nerve cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm; and / or
    所述神经细胞小室的厚度为10～200μm，优选为50～150μm，最优选为100μm。The thickness of the nerve cell compartment is 10 to 200 μm, preferably 50 to 150 μm, and most preferably 100 μm.
  3. 根据权利要求1或2所述的微流控芯片，其特征在于，所述癌细胞小室的长度为1～10mm，优选为2～6mm，最优选为4mm；The microfluidic chip according to claim 1 or 2, wherein the length of the cancer cell compartment is 1 to 10 mm, preferably 2 to 6 mm, and most preferably 4 mm;
    所述癌细胞小室的宽度为1～10mm，优选为1～3mm，最优选为2mm；和/或The width of the cancer cell compartment is 1 to 10 mm, preferably 1 to 3 mm, and most preferably 2 mm; and / or
    所述癌细胞小室的厚度为10～200μm，优选为50～150μm，最优选为100μm。The thickness of the cancer cell compartment is 10 to 200 μm, preferably 50 to 150 μm, and most preferably 100 μm.
  4. 根据权利要求1至3中任一项所述的微流控芯片，其特征在于，所述微通道的长度为100～500μm，优选为100～300μm，最优选为200μm；The microfluidic chip according to any one of claims 1 to 3, wherein a length of the microchannel is 100 to 500 μm, preferably 100 to 300 μm, and most preferably 200 μm;
    所述微通道的宽度为5～50μm，优选为5～20μm，最优选为10μm；和/或The width of the microchannel is 5-50 μm, preferably 5-20 μm, and most preferably 10 μm; and / or
    所述微通道的厚度为5～50μm，优选为5～20μm，最优选为10μm。The thickness of the microchannel is 5 to 50 μm, preferably 5 to 20 μm, and most preferably 10 μm.
  5. 根据权利要求1至4中任一项所述的微流控芯片的制备方法，其特征在于，所述方法包括以下步骤：The method for preparing a microfluidic chip according to any one of claims 1 to 4, wherein the method includes the following steps:
    (1)设计微流控芯片，打印掩膜版；(1) Design microfluidic chip and print mask version;
    (2)利用光刻技术制备模板；(2) using a photolithography technique to prepare a template;
    (3)在模板上浇筑PDMS预聚物，加热，从掩膜版上剥下所述PDMS得到所述微流控芯片。(3) Pouring PDMS prepolymer on the template, heating, and peeling the PDMS from the mask to obtain the microfluidic chip.
  6. 根据权利要求5所述的方法，其特征在于，所述步骤(3)中，所述PDMS预聚物中，PDMS与交联剂的质量比为5～20:1，优选为5～15:1，最优选为10:1。The method according to claim 5, characterized in that in the step (3), in the PDMS prepolymer, a mass ratio of PDMS to a crosslinking agent is 5 to 20: 1, preferably 5 to 15: 1, most preferably 10: 1.
  7. 根据权利要求5或6所述的方法，其特征在于，所述步骤(3)中，所述加热温度为50～150℃，优选为50～100℃，最优选为80℃。The method according to claim 5 or 6, wherein in the step (3), the heating temperature is 50 to 150 ° C, preferably 50 to 100 ° C, and most preferably 80 ° C.
  8. 一种高通量药物筛选平台，其特征在于，所述平台包括：A high-throughput drug screening platform is characterized in that the platform includes:
    权利要求1～4中任一项所述的微流控芯片；和The microfluidic chip according to any one of claims 1 to 4; and
    高内涵成像系统。High-content imaging system.
  9. 权利要求1～4中任一项所述的微流控芯片或权利要求8所述的高通量药物筛选平台在制备药物筛选产品尤其是高通量药物筛选产品中的应用。Application of the microfluidic chip according to any one of claims 1 to 4 or the high-throughput drug screening platform according to claim 8 in the preparation of a drug screening product, especially a high-throughput drug screening product.
  10. 根据权利要求9所述的应用，其特征在于，所述药物为神经-肿瘤微环境相关药物。The application according to claim 9, wherein the drug is a neuro-tumor microenvironment-related drug.
  11. 一种药物筛选方法，其特征在于，所述方法采用：A drug screening method, characterized in that the method uses:
    如权利要求1～4中任一项所述的微流控芯片；和/或The microfluidic chip according to any one of claims 1 to 4; and / or
    如权利要求8所述的高通量药物筛选平台。The high-throughput drug screening platform of claim 8.
  12. 一种细胞共培养装置，其特征在于，所述装置包括权利要求1～4中任一项所述的微流控芯片。A cell co-culture device, characterized in that the device comprises the microfluidic chip according to any one of claims 1 to 4.
  13. 一种细胞共培养方法，其特征在于，所述方法采用如权利要求1～4中任一项所述的微流控芯片。A cell co-culture method, characterized in that the method uses the microfluidic chip according to any one of claims 1 to 4.
  14. 一种细胞识别平台，其特征在于，所述平台包括权利要求1～4中任一项所述的微流控芯片；A cell recognition platform, characterized in that the platform comprises the microfluidic chip according to any one of claims 1 to 4;
    优选地，所述细胞识别为细胞-细胞识别和/或细胞-微环境识别。Preferably, the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
  15. 一种细胞识别方法，其特征在于，所述方法采用权利要求1～4中任一项所述的微流控芯片；A cell identification method, characterized in that the method uses the microfluidic chip according to any one of claims 1 to 4;
    优选地，所述细胞识别为细胞-细胞识别和/或细胞-微环境识别。Preferably, the cell recognition is cell-cell recognition and / or cell-microenvironment recognition.
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