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Jan 2020

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A Novel and Robust Single-cell Trapping Method on Digital Microfluidics
一种新颖、可靠的数字微流控单细胞捕获方法   

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Abstract

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

Keywords: Single-cell (单细胞), Digital Microfluidics (数字微流控), Microstructures (显微结构), Cell Culture (细胞培养), Drug Screening (药物筛选)

Background

Cellular heterogeneity within a large cell population is common. Individual cell analysis will provide more accurate information about the cell-to-cell variation masked by the stochastic average in bulk analysis. Single cell culture, which is the first step of individual cell analysis, becomes very important.

Flow cytometry is a commonly used method for single cell analysis. However, the requisition of large cell numbers (more than 10,000) hurdled its applications in the research of limited precious samples. With the merits of low sample consumption, low analysis cost, microfluidics has been pursued by many scientists, especially in the research field of limited sample acquisition and expensive reagents needed, such as cell-based drug screening. Digital microfluidics, which utilizes electric signal to manipulate individual droplet has shown its promising in cell-culture related research. However, it is difficult to realize single cell capture on a flat electrode on digital microfluidics considering several hundred nanoliter sized droplets. Although several researchers reported that they can isolate single cell on digital microfluidics, the drawbacks hindered their further applications. For example, Gidrol’s group realized single cell isolation by adjusting the concentrations of cell suspensions (Rival et al., 2014). However, the single-cell isolation efficiency was quite low. Lammertyn’s group fabricated a series of micro sized hydrophilic micropatches on an electrode for single cell trapping (Witters et al., 2011). However, the multiple hydrophilic patches resulted in higher droplet transportation voltage, which will not only shorten the chip lifetime, but also cause potential damage to cells.

In our recent work, we developed a single cell culture method for primary tumor drug screening based on microfabrication and digital microfluidic technologies (Zhai et al., 2020). In the work, 3D microstructures were engineered on digital microfluidic chips for single-cell isolation and long-time culture. With novel combination of medium oil and additives, the droplet actuation voltage was lowered to 36 V, 4 times lower than normally used. Following, breast cancer drug screening was run on-chip with the designed microstructures showing consistent results as in conventional 96-well plates.

In the present protocol, we provide detailed steps for this single cell trapping method, including experimental details to perform single cell culture with the digital microfluidic system.

Materials and Reagents

For 3D microstructures fabrication on chip

  1. Adhesive tape (3M, catalog number: 1183 )
  2. Chromium patterned bottom plate (Changsha Shaoguang Chromium Edition Company, China)
  3. ITO top plate (Zhuhai Kaivo Optoelectronic Technology Company, China)
  4. Isopropanol, abbreviate as IPA (Fisher Chemical, catalog number: 182060 )
  5. Acetone (Fisher Chemical, catalog number: 183005R )
  6. Milli-Q water (18.2 MΩ)
  7. SU-8 3010 (MicroChem, catalog number: 17120963 )
  8. SU-8 2050 (MicroChem, catalog number: 17070500 )
  9. SU-8 developer (MicroChem, catalog number: 18090763 )
  10. Amorhpous Fluoroplastics Solution (6% Teflon solution) (Chemours, catalog number: 1706ES0014 )
  11. N2 (Junyu company, China)
  12. FC-40 (3M, catalog number: 20051 )

For cell suspensions preparation

  1. Pipette tips10 μl (Eppendorf, catalog number: 0 224930000 )
  2. Pipette tips 200 μl (Eppendorf, catalog number: 0 22491296 )
  3. Silicone oil (Clearco, CAS number: 107-51-7 )
  4. Breast cancer cell lines MDA-MB-231 (ATCC, catalog number: BNCC245095 )
  5. RPMI 1640x basic medium (Gibco, catalog number: 11875-093 )
  6. Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106 )
  7. Trypsin (Gibco, catalog number: 25200-056 )
  8. Pluronic® F-127, abbreviate as F127 (Sigma-Aldrich, CAS number: 9003-11-6 )
  9. Cisplatin (Sigma, catalog number: P4394 )
  10. EthD-1 (Thermo Fisher Scientific, catalog number: E1169 )
  11. Cell Counting Kit-8 (CCK-8) (Dojindo, Shanghai)

For single cell trapping on chip

  1. Pipette tips10 μl (Eppendorf, catalog number: 0 224930000 )
  2. Pipette tips 200 μl (Eppendorf, catalog number: 0 22491296 )
  3. Silicone oil (Clearco, CAS number: 107-51-7 )

Equipment

  1. Plasma cleaner (ABM, model: MR-200S )
  2. UV exposure (ABM, model: ABM-350J )
  3. Hot plate (Qiangyuan Instrument, model: BX-1 )
  4. Laser cutting machine (ZKJ Laser, Shang Hai)
  5. Clean bench (Lansi Purification, model: LANSI-GZ-01 )
  6. Cell incubator (Thermo Fisher, model: Steri-Cycle i160 CO2 incubator )
  7. Pipette (Eppendorf, Research plus single channel adjustable range pipette)
  8. Microscope (Olympus, model: BX63F )
  9. Cellometer (Mini, Dakewe, China)
  10. Microplate reader (Thermo Fisher, ID number: IED0501003M )
  11. Spin coater (KW-4A, SETCAS Electronics Company, China)

Software

  1. Auto CAD 2017 (Autodesk company)
  2. ImageJ (NIH government)

Procedure

The single cell trapping protocol can be divided into 4 parts: 3D microstructure design, 3D microstructures construction on chip, cell suspensions preparation, and oil film with surfactant for single cell trapping on chip. The final fraction is done by the digital microfluidic system. The detailed protocols for each part are as following:


  1. 3D microstructure design
    We use AutoCAD to design the microstructures. The microstructures include repetitive structure arrays (Figure 1). The yellow frame in Figure 1 shows the single structure, which consisted of four walls (rectangular frames), each one with a width of 7 μm, a length of 20 μm, and a small gap of 2.5 μm (Figure 1, right) between the ends of each wall, forming a semi-closed well between the walls. The designed microstructures were used to produce a mask for the following photolithography experiments.


    Figure 1. The designed structures for single cell culture with Auto CAD

  2. For 3D microstructures fabrication on chip
    The DMF chip is composed of three parts: the bottom plate, the top plate, and the conductive adhesive tape as spacer to connect the top plate and bottom plate. We used autoCAD to design the electrode patterns. Then chromium electrodes (Cr) were patterned on glass plates by and purchased from Changsha Shaoguang Chromium Edition Company, China. ITO top plates were purchased from Zhuhai Kaivo Optoelectronic Technology Company, China. Figure 2 showed the process for the bottom and top plates fabrication and the assembling of bottom plate and top plate with spacer.


    Figure 2. The process for the bottom (A-D) and top plate (E-F) fabrication. (G) The assembling of bottom plate and top plate with spacer to form a DMF chip

    The detailed protocols are as following:
    1. Coat 10 μm SU-8 layer on the bottom plate as the dielectric layer.
      1. Plasma treatment for the designed chromium patterned bottom plate for 1 min (The plate is placed in the chamber of plasma cleaner equipment. First, the plate is treated with vacuum and then oxygen. Then the surface of the plate will be ionized. This process is called plasma treatment. The plasma treated plate will promote the bonding between SU-8 and plate. Plasma treatment is a very common used technique in the microfabrication field.).
      2. Pour SU-8 3010 (4 ml) on the bottom plate to cover about 1/3 area of the chip.
      3. Spin coat at 500 rpm, 5 s and sequentially 3,000 rpm, 30 s with spin coater (The procedure is as follows: excess SU-8 solution was poured to the glass substrate, which is rotated at high speed to spread the SU-8 solution by centrifugal force until the solution spins off the edges of the glass substrate and the desired thickness of the SU-8 film is achieved).
      4. Bake the chip for 3 min at 65 °C, 5 min at 95 °C on hot plate.
      5. Precise patterning of the dielectric layer with mask aligner via UV exposure for 18.5 s.
      6. Post-bake the chip at 65 °C for 3 min and 95 °C for 3 min on hot plate.
      7. Develop the chip with SU-8 developer for 1 min (The plate was immersed in the SU-8 developer solution. The transparent parts on the mask after UV exposure will help UV light irradiate the plate directly and strengthen the bonding between SU-8 and the plate. After developing, the pattern of the transparent parts on the mask will be printed as SU-8 film on the plate, while the pattern of the opaque parts on the mask will not be printed and washed off.).
    2. Coat 60 μm thickness SU-8 on the chip as fences to prevent droplets from drifting.
      1. Plasma treatment for the bottom plate for 1 min.
      2. Pour SU-8 2050 (4 ml) on the bottom plate to cover about 1/3 area of the chip.
      3. Spin coat at 500 rpm, 5 s and sequentially 2,500 rpm, 30 s with spin coater.
      4. Bake the chip for 3 min at 65 °C, 10 min at 95 °C on hot plate.
      5. Precise patterning with mask aligner via UV exposure for 13.5 s.
      6. Post-bake the chip at 65 °C for 3 min and 95 °C for 5 min on hot plate.
      7. Develop the chip with SU-8 developer for 5 min.
    3. Coat 10 μm thickness SU-8 layer as a microstructure array to perform single-cell culture.
      1. Plasma treatment for the bottom plate for 1 min.
      2. Pour SU-8 3010 (4 ml) on the bottom plate to cover about 1/3 area of the chip.
      3. Spin coat at 500 rpm, 5 s and sequentially 3,000 rpm, 30 s with spin coater.
      4. Bake the chip for 3 min at 65 °C, 5 min at 95 °C on hot plate.
      5. Precise patterning with mask aligner via UV exposure for 18.5 s.
      6. Post-bake the chip at 65 °C for 3 min and 95 °C for 3 min on hot plate.
      7. Develop the chip with SU-8 developer for 1 min.
    4. Coat Teflon on both the bottom plate and top plate.
      1. Prepare 0.5%Teflon solution 100 ml (use FC-40 to dilute 6% Teflon solution to 0.5%).
      2. Wash the bottom plate and top plate with 5 ml IPA and 5 ml MilliQ-water.
      3. Blow dry the bottom plate and top plate with N2.
      4. Dry off the top plate and bottom plate with hot plate at 100 °C.
      5. Cool down the top plate and bottom plate at the room temperature.
      6. Use pipette to transfer adequate amount of 0.5% teflon solution (about 1 ml) to cover the bottom plate and top plate.
      7. Spin coat the bottom plate and top plate for 500 rpm, 10 s and sequentially 1,000 rpm, 60 s with spin coater.
      8. Bake the bottom plate and top plate for 10 min at 185 °C on hot plate.

    The SEM image result for the 3D microstructures is shown in Figure 3.


    Figure 3. The SEM image result for the designed 3D microstructures

  3. Chip assembly
    The DMF chip consists of the bottom plate, a spacer (made of adhesive tapes) to connect the two plates, and a top plate, as shown in the insert real image of Figure 4. The bottom plate is a main part of the DMF chip. A series of chromium electrodes are designed and fabricated on a glass plate, which is used for droplet operation by charging the electrodes or not. We also call it as bottom plate. A 1.5 mm diameter hole is drilled on the top plate by laser cutting machine for sample loading (the inserted image in Figure 4). The scheme of the chip pattern is shown in Figure 4, including sample loading position, the electrodes with microstructures for cell culture and other electrodes aid for the sample moving to the cell culture spots. All the cell culture spots in Figure 4 contain the semi closed well system illustrated by the Figure 3 SEM image. The grey droplet in Figure 4 illustrates the travel of a drug or cell culture suspension from the pipette tip to one of the cell culture compartments.


    Figure 4. The scheme of the chip pattern and the image of the assemble chip

  4. Cell suspensions preparation
    1. Discard the medium when the MDA-MB-231 cells grow to 90% density in the 6-well plates.
    2. Add 0.5 ml trypsin to the well plate and shake to wet the whole well, then discard the trypsin incubate at 37 °C for 1 min in the cell incubator.
    3. Add 0.5 ml RPMI 1640 medium (supplement with 10% FBS) to the well plate.
    4. Use a pipette to transfer the cell suspensions to 1.5 ml sterile centrifuge tube.
    5. Count the cell concentration with Cellometer and adjust the cell concentration to 8 x 105 cells/ml with RPMI 1640 medium.
    6. Add F127 to the MDA-MB-231 cell suspension with the percent of 0.01%.

  5. Oil film with surfactant for single cell trapping on chip
    1. Fill up the space between the bottom and top plates of the chip with oil via a pipette.
    2. Pipette 0.6 μl droplets (8 x 105 MDA-MB-231 cells/ml) containing 0.01% Pluronic F127 into the holes and move the droplets of cells to the virtual chambers by charging the adjacent electrodes sequentially (Figure 5 showed the DMF system. The electronic control system was used to control the charge of the DMF chip. Each electrode is connected to a contact electrode pad patterned at the edge of the DMF chip through chromium lines patterned on chip, and further connected to the control electronics panel by jumper wires connector. A power supply is connected to the contro electronics. Physical relays on the control board are used as the switches to turn on or turn off the charging of each electrode. The commands are sent from a computer software via Bluetooth to the control electronics). All the operations were carried out in clean bench.
    3. Place the chip in a humidified incubator (37 °C, 5% CO2) for a long-time culture (24 h).


      Figure 5. System setup. The DMF system was consisted of four parts: a DMF chip, electronic control system (power supply, signal generator, PCB board and connectors), a self-written control software and a fluorescence microscope.

      Figure 6 is an example image of a compartment of the "cell culture spots" in Figure 4. Each circle is a cell in this image. The restriction of the 3D microstructures beneath each droplet would force the droplet to form a curved interface due to the interfacial tension, which promotes the single cell trapping. The cells sit in the semi closed well, as shown in Figure 6.


      Figure 6. The image result for single cell culture with the designed 3D microstructures

  6. Example on breast cancer drug toxicity test
    Drug toxicity test on DMF chip
    1. Fill up the space between the bottom and top plates of the chip with oil using a pipette.
    2. Pipette 0.6 μl droplets (8 x 105 MDA-MB-231 cells/ml) containing 0.01% Pluronic F127 and 2 μM EthD-1 and cisplatin solution (0.6 μl) in a series of concentrations into the holes and move to the virtual chambers by charging the adjacent electrodes sequentially and mix on the DMF chip. All the operations were carried out on a clean bench.
    3. Place the chip in a humidified incubator (37 °C, 5% CO2) for a long-term culture (24 h).
    4. Measure the red fluorescence of the cells under the Microscope.

    Drug toxicity test off-chip
    1. Seed MDA-MB-231 cells (1.0 x 104 cells/well, 100 μl RPMI 1640 medium supplied with 10% FBS) in a 96-well plate for 12 h.
    2. Discard the medium and pipette a series of cisplatin concentrations (100 μl) with each concentration in triplicates to the 96-well plate and culture for 24 h.
    3. Add 10 μl CCK-8 solution to each well and incubate for 0.5 h.
    4. Measure 450 nm absorbance of each well using a microplate reader.
    The cisplatin drug toxicity result for MDA-MB-231 cells is shown in Figure 7.


    Figure 7. The Cisplatin (Cis) drug toxicity test on MDA-MB-231 breast cancer cells. A. The on-chip fluorescence image results for 0 μM, 10 μM Cis-treated MDA-MB-231 cells for 24 h. B. The drug toxicity test results for Cis-treated MDA-MB-231 cells on /off-chip (96-well plate).

Acknowledgments

This work was supported by Macau Science and Technology Development Fund (FDCT) [FDCT110/2016/A3, FDCT 0053/2019/A1, AMSV SKL Fund]; University of Macau [MYRG2017-00022-AMSV, MYRG2018-00114-AMSV].

Competing interests

The authors declare that they have no conflict of interest.

References

  1. Rival, A., Jary, D., Delattre, C., Fouillet, Y., Castellan, G., Bellemin-Comte, A. and Gidrol, X. (2014). An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR. Lab Chip 14(19): 3739-3749.
  2. Witters, D., Vergauwe, N., Vermeir, S., Ceyssens, F., Liekens, S., Puers, R. and Lammertyn, J. (2011). Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications. Lab Chip 11(16): 2790-2794.
  3. Zhai, J., Li, H., Wong, A. H. H., Dong, C., Yi, S., Jia, Y., Mak, P. I., Deng, C. X. and Martins, R. P. (2020). A digital microfluidic system with 3D microstructures for single-cell culture. Microsystems Nanoengineering 6(1): 6.

简介

[摘要]由于细胞的异质性,在批量分析方法中,尤其是在对患者的原发性肿瘤活检样品进行分析时,会平均各个细胞之间的差异。为了深入了解原发性肿瘤中的细胞间差异,对细胞数量有限的单细胞培养和分析提出了很高的要求。鉴于其小反应量的要求,微流体一直是解决该问题的最佳平台。数字微流体,它利用一个电信号操纵单个液滴小号显示PROMIS ê在细胞培养物与易控制。在这项工作中,我们通过在芯片上制造3D微结构以形成半封闭微孔,在数字微流控平台上实现单细胞捕获。通过这种设计,隔离的单个单元可以占用30 x 30阵列的20%。我们还使用低蒸发硅油和氟化表面活性剂来降低液滴驱动电压并防止液滴蒸发,同时在长期培养(24 h)中允许细胞呼吸。如本协议所示,在数字微流控技术上进行单细胞捕获的主要步骤包括3D微结构设计,芯片上3D微结构构建以及带有表面活性剂的油膜,用于芯片上单细胞捕获。


[背景]大的细胞群中的细胞异质性是常见的。单个细胞分析将提供有关大量分析中随机平均值所掩盖的细胞间变化的更准确信息。作为单个细胞分析的第一步,单细胞培养变得非常重要。

流式细胞术是单细胞分析的常用方法。然而,大细胞数量(超过10,000个)的需求限制了其在有限珍贵样品研究中的应用。具有低样品消耗,低分析成本的优点,许多科学家一直在追求微流体技术,特别是在样品采集受限和需要昂贵试剂(例如基于细胞的药物筛选)的研究领域。利用电信号操纵单个液滴的数字微流控技术在细胞培养相关研究中显示出了其前途。然而,这是难以实现的考虑几百纳升大小的液滴的数字微流体在一个平面电极单细胞捕获小号。尽管一些研究人员报告说他们可以在数字微流控技术上分离单个细胞,但其缺点阻碍了它们的进一步应用。例如,Gidrol的组通过调整细胞悬浮液的浓度来实现单细胞分离(对手等人,2014) 。但是,单电池隔离效率非常低。Lammertyn的组制作的系列微尺寸的亲水性的micropatches对单个CE电极LL俘获(威特斯等人,2011) 。然而,多个亲水性贴剂导致更高的液滴输送电压,这不仅会缩短芯片寿命,而且还会对细胞造成潜在的损害。

在我们最近的工作中,我们开发了原发肿瘤药物筛选单个细胞培养方法基于微细加工和数字微流体技术(翟等人,20 20 )。在这项工作中,在数字微流控芯片上设计了3D微结构,用于单细胞分离和长期培养。通过新型的中油和添加剂组合,液滴的驱动电压降低到36 V,比通常使用的电压低4倍。随后,乳腺癌药物筛选在芯片上进行,设计的微结构显示出与常规96孔板相同的结果。

在本协议中,我们为这种单细胞捕获方法提供了详细的步骤,包括使用数字微流系统进行单细胞培养的实验细节。

关键字:单细胞, 数字微流控, 显微结构, 细胞培养, 药物筛选


材料和试剂

用于芯片上的3D微结构制造

1.胶带(3M,目录号:1183 )     
2. Ç hromium图案化的底板(长沙韶光铬版公司,中国)     
3. ITO顶板(中国珠海凯沃光电技术有限公司)     
4. I异丙醇,缩写为IPA(Fisher Chemical,目录号:182060)     
5.丙酮(Fisher化学公司,Ç atalog数:183005R)                   
6. Milli-Q水(18.2MΩ)     
7. SU-8 3010(MicroChem ,目录号:17120963)     
8. SU-8 2050(MicroChem ,目录号:17070500)     
9. SU-8显影剂(MicroChem ,目录号:18090763)     
10. Amorhpous氟塑料溶液(6%Ť EFLON溶液)(Chemours,目录号:1706ES0014) 
11. N 2 (中国骏宇公司) 
12. FC-40(3M,目录号:20051) 
用于细胞悬浮液的制备

1.细胞培养6孔板(Thermo Fisher Scientific,目录号:172931)     
2.细胞培养9个6孔板(Corning,目录号:3599 )     
3. 1.5 ml离心管(Thermo Fisher Scientific,目录号:339650)     
4.乳腺癌细胞系MDA-MB-231(ATCC ,目录号:BNCC245095 )      5. RPMI 1640x基本介质(Gibco,目录号:11875-093)      6.胎牛血清(FBS)(Gibco,目录号:10270-106)     
7.胰蛋白酶(Gibco,目录号:25200-056)     
8.普朗尼克® F127 ,简称为F127 (西格玛- Aldrich公司,CAS号:9003-11-6 )     
9.顺铂(Sigma ,目录号:P4394)     
10. EthD-1(Thermo Fisher Scientific,目录号:E1169 ) 
11.细胞计数试剂盒8(CCK-8)(上海同仁堂) 
用于芯片上的单细胞捕获

1.移液器吸头10μl (Eppendorf,目录号:0224930000)     
2.移液器吸头200μl (Eppendorf,目录号:022491296)     
3.硅油(Clearco ,CAS号:107-51-7)     
设备

等离子清洁器(ABM,型号:MR-200S)
紫外线(ABM,型号:ABM -350J )
热板(墙垣我nstrument,型号:BX-1 )
激光切割机(ZKJ Laser,上海)
洁净工作台(蓝丝P urification ,型号:蓝丝-GZ-01 )
细胞培养箱(热˚F isher ,型号:免缝-CYCLE I160 CO 2培养箱)
移液器(Eppendorf ,Research plus单通道可调范围移液器)
显微镜(奥林巴斯,型号:BX63F )
细胞仪(Mini,达克威,中国)
酶标仪(Thermo F垫圈,ID号:IED0501003M )
旋涂机(KW-4A ,SETCAS电子公司,中国)
软件

Auto CAD 2017 (Autodesk公司)
图片J(NIH政府)
程序

单细胞捕获方案可分为4个部分:3D微结构设计,芯片上3D微结构构建,细胞悬浮液制备以及带有表面活性剂的油膜,用于单细胞捕获。最后部分由数字微流体系统完成。每个部分的详细协议如下:

3D微结构设计
我们使用AutoCAD设计微结构。微观结构包括重复结构阵列(图1)。图1显示了黄色帧中的单一结构,其包括四个壁(矩形框或多个),每一个为7的宽度微米,20的长度微米,和2.5的小间隙微米(˚F igure 1,右)在每个墙的末端之间,在墙之间形成一个半封闭的井。设计的微结构用于生产用于以下光刻实验的掩模。

D:\ Reformatting \ 2020-8-3 \ 2003144--1513贾彦伟870615 \ Figs jpg \ figure1.jpg

图1.使用Auto CAD进行单细胞培养的设计结构

用于芯片上的3D微观结构制造
DMF芯片由三部分组成:底板,顶板和导电胶布,作为连接顶板和底板的垫片。我们使用autoCAD设计电极图案。然后,铬电极(Cr)由中国长沙韶光铬版公司购买,并在玻璃板上构图。ITO顶板购自中国珠海凯沃光电技术公司。图2显示了底板和顶板的制造过程以及带有垫片的底板和顶板的组装过程。

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图2 。底部(AD)和顶部板(EF)的制造过程。(G)用隔板组装底板和顶板以形成DMF芯片

详细协议如下:

涂层10微米SU-8层上的底板作为介电层。
对设计的铬制图案底板进行等离子处理1分钟(将板放置在等离子清洁器设备的腔室中。首先,先对板进行真空处理,然后再进行氧气处理,然后将板表面电离。此过程是称为等离子体处理。该等离子体处理的板将促进SU-8和板。之间的键合等离子处理是一种非常常见的微细加工领域中使用的技术。)。
倾SU-8 3010 (4米升)在所述底板上以覆盖约1/3面积的芯片。
用旋涂机以500 rpm,5 s和依次3,000 rpm,30 s的速度旋涂(步骤如下:将过量的SU-8溶液倒入玻璃基板,然后高速旋转以分散SU-8溶液通过离心力直到溶液旋转掉玻璃基板和SU-8膜的所需厚度的边缘实现)。
在65 °C下将芯片烘烤3分钟,在热板上在95 °C下烘烤5分钟。
通过紫外线照射18.5 s,用掩模对准器对介电层进行精确的图案化。
在热板上将芯片在65 °C下烘烤3分钟,然后在95 °C下烘烤3分钟。
用SU-8显影剂将芯片显影1分钟(将板浸入SU-8显影剂溶液中。UV曝光后,掩模上的透明部分将有助于UV光直接照射板,并增强SU-8与显影剂之间的结合力该板。显影后,将透明部件的掩模上的图案将被打印为在板SU-8膜,而掩模上的不透明部分的图案将不被打印并洗涤掉。)。
涂层60微米厚度SU-8的芯片作为围栏以防止液滴从漂流。
底板的等离子体处理1分钟。
倾SU-8 2050 (4米升)在所述底板上以覆盖约1/3面积的芯片。
使用旋涂机以500 rpm,5 s的速度旋涂,随后以2500 rpm,30 s的速度旋涂。
在65 °C下将芯片烘烤3分钟,在95 °C下在热板上烘烤10分钟。
通过紫外线曝光13.5 s,使用掩模对准器进行精确图案化。
在热板上将芯片在65 °C下后烤3分钟,然后在95 °C下后烤5分钟。
用SU-8开发人员将芯片开发5分钟。
将10μm厚的SU-8层涂覆为微结构阵列,以进行单细胞培养。
底板的等离子体处理1分钟。
倾SU-8 3010 (4米升)在所述底板上以覆盖约1/3面积的芯片。
使用旋涂机以500 rpm,5 s的速度旋涂,然后以3,000 rpm,30 s的速度依次旋涂。
在65 °C下将芯片烘烤3分钟,在热板上在95 °C下烘烤5分钟。
通过掩模对准器通过UV曝光18.5 s进行精确图案化。
在热板上将芯片在65 °C下烘烤3分钟,然后在95 °C下烘烤3分钟。
用SU-8开发人员将芯片开发1分钟。
在底板和顶板上都涂上聚四氟乙烯。
制备0.5%的碲氟里昂溶液百米升(使用FC-40稀释6%特氟隆溶液至0.5% )。
W¯¯灰底板和顶板用5米升IPA和5米升的MilliQ -水。
B用N 2将底板和顶板低干。
d RY切断顶板和底板用H OT板在100 ℃下。
Ç OOL向下吨他顶板和底板,在室温下。
使用移液管转移的足够量的0.5%吨EFLON溶液(约1米升),以覆盖所述底板和顶板。
小号针外套吨他底板和顶板为500rpm下,10秒,并依次以1000rpm,60秒用旋涂机。
在185 °C的热板上烘烤底板和顶板10分钟。
3D微观结构的SEM图像结果如图3所示。

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图3 。设计的3D微结构的SEM图像结果

                                                                                                   芯片组装
Ť他DMF芯片由底板,间隔的(制成胶带),以连接两块板,以及顶板,如图的插入实像˚F igure 4.底板是的主要部分DMF芯片。一系列的铬电极的设计和在玻璃板上,其用于液滴操作制成由充电电极或没有吨。我们也称其为底板。1.5毫米直径的孔被钻出在顶板通过激光切割机对样品加载(图插入的图像4 )。芯片图案的示意图如图4所示,包括样品的装载位置,具有用于细胞培养的微结构的电极和其他电极,以帮助样品移至细胞培养点。图4中的所有细胞培养点均包含图3 SEM图像所示的半封闭孔系统。图4中的灰色液滴表示药物或细胞培养悬液从移液器吸头到细胞培养隔室之一的行程。
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图4 。芯片图案方案与组装芯片图像

                                                                                                  细胞悬液制备
1. d iscard的介质瓦特母鸡的MDA-MB-231细胞生长至90%的密度在6孔板。     
2.添加0.5毫升胰蛋白酶到孔板振摇润湿全井,吨母鸡丢弃胰蛋白酶孵育37 ℃下在细胞培养箱1分钟。                                                                                       
3.向孔板中加入0.5 ml RPMI 1640培养基(补充10%FBS)。                                                                                       
4.使用一个p ipette到细胞悬浮液转移到1.5毫升无菌的离心管中。                                                                                       
5. Ç指望。2-15细胞浓度与Cellometer并调节细胞浓度至8×10 5个细胞/ ml用RPMI 1640培养基。                                                                                       
6.将F127以0.01%的百分比添加到MDA-MB-231细胞悬液中。                                                                                       
                                                                                                   具有表面活性剂的油膜,可将单细胞捕获在芯片上
1. ˚F生病了空间B切口白内障手术挽下部,并用石油芯片顶部板通过移液管。           
2. P ipette 0.6微升液滴(8×10 5含0.01%的Pluronic F127到孔MDA-MB-231细胞/ ml)和移动细胞的液滴通过顺序充电相邻电极到虚拟室(图5显示了DMF系统:使用电子控制系统控制DMF芯片的电荷,每个电极通过在芯片上构图的铬线连接到在DMF芯片边缘构图的接触电极焊盘,并进一步连接到控制电子面板通过跨接线连接器。将电源连接到控制电子设备。控制板上的物理继电器用作打开或关闭每个电极充电的开关。命令从计算机软件通过蓝牙发送到电子控制装置)。所有操作均在洁净台上进行。           
3. P花边吨他芯片在加湿培养箱(37℃,5%CO 2 )长期-t IME立方米lture (24小时)。           
D:\ Reformatting \ 2020-8-3 \ 2003144--1513贾彦伟870615 \ Figs jpg \ figure5.jpg

图5 。系统设置。DMF系统由四部分组成:DMF芯片,电子控制系统(电源,信号发生器,PCB板和连接器),自写控制软件和荧光显微镜。

图6是图4中“细胞培养点”的隔室的示例图像。每个圆圈是此图像中的一个单元。每个界面下方的3D微结构的限制会由于界面张力而迫使该界面曲面形成弯曲界面,从而促进了单细胞捕获。单元格位于半封闭井中,如图6所示。

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图6 。具有设计的3D微结构的单细胞培养的图像结果

                                                                      例如在b reast抗癌药毒性试验
DMF芯片的药物毒性测试

                                                          用移液器在机油的底部和顶板之间充满油。
                                                          吸管0.6微升液滴(8×10 5 MDA-MB-231细胞/ ml)含有0.01%的Pluronic F127和2 μM EthD-1和氯氨铂溶液(0.6微升)在一系列浓度的插入孔中并移动到虚拟室通过顺序充电相邻电极并在DMF芯片上混合。所有操作均在干净的工作台上进行。
                                                          将芯片放在潮湿的培养箱中(37°C,5%CO 2 )进行长期培养(24 h)。
                                                          在显微镜下测量细胞的红色荧光。


药物毒性试验片外

                                                          种子MDA-MB-231细胞(1.0 X 10 4细胞/孔,100微升RPMI 1640培养基中增刊我é d有10%FBS )的96孔板中12小时。
                                                          丢弃培养基,移液管的一系列顺铂浓度(的100微升)以一式三份各浓度小号到96孔板并培养24小时。
                                                          添加10 μ升CCK-8溶液以每孔并孵育0.5小时。
                                                          测量450nm处吸光度每孔的用一个微板读数器。
在被示出的MDA-MB-231细胞顺铂药物毒性结果˚F igure 7。

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图7.对MDA-MB-231乳腺癌细胞的顺铂(Cis)药物毒性测试。A.在芯片上的荧光为0的图像效果μM ,10个μM顺式处理的MDA-MB-231细胞24小时。B.芯片上/芯片外(96孔板)的经Cis处理的MDA-MB-231细胞的药物毒性测试结果。

致谢

这项工作得到了澳门科技发展基金(FDCT)[FDCT110 / 2016 / A3,FDCT 0053/2019 / A1,AMSV SKL基金]的支持;澳门大学[MYRG2017-00022-AMSV,MYRG2018-00114-AMSV]。

利益争夺

作者宣称他们没有利益冲突。

参考文献

Rival,A.,Jary ,D.,Delattre,C.,Fouillet ,Y.,Castellan,G.,Bellemin- Comte,A.和Gidrol ,X.(2014)。基于EWOD的微流控芯片,用于单细胞分离,mRNA纯化和后续的多重qPCR。实验芯片14(19):3739-3749。
威特斯,D.,Vergauwe ,N.,Vermeir ,S.,Ceyssens ,F.,Liekens ,S.,Puers ,R。和Lammertyn ,J。(2011)。电介质上电润湿数字微流控芯片的生物功能化,用于基于单元的小型化应用。实验芯片11(16):2790-2794。
翟,J.,李,H.,黄,AH H.,侗,C.,彝,S.,贾,Y.,麦,P一,邓C. X.和马丁斯,RP(2020 )。具有3D微结构的数字微流体系统,用于单细胞培养。微系统纳米工程6(1):6。
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引用:Zhai, J., Li, H., Wong, A., Dong, C., Yi, S., Jia, Y., Mak, P., Deng, C. and Martins, R. (2020). A Novel and Robust Single-cell Trapping Method on Digital Microfluidics. Bio-protocol 10(19): e3769. DOI: 10.21769/BioProtoc.3769.
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