微流体设备中的电场诱导神经前体细胞分化
Summary
在这项研究中，我们提出了一个单纯由微流系统中直接电流（DC）脉冲刺激诱导的神经干细胞和祖细胞（NPC）分化的协议。
Abstract
生理电场 （EF） 在细胞迁移、分化、分裂和死亡中起着至关重要的作用。本文描述了一个利用显微镜进行长期细胞分化研究的微流体细胞培养系统。微流体系统由以下主要部件组成：光学透明电控芯片、透明钛-锡氧化物 （ITO） 加热器、培养物介质灌装泵、电源、高频功率放大器、EF 多路复用器、可编程 X-Y-Z 电动舞台以及配备数码相机的倒相对比显微镜。微流体系统有利于简化整体实验设置，进而有利于试剂和样品的消耗。这项工作涉及由直接电流（DC）脉冲刺激诱导的神经干细胞和祖细胞（NPC）的分化。在干细胞维持介质中，小鼠NC（mNPC）在直流脉冲刺激后分化成神经元、星形细胞和寡头细胞。结果表明，简单的直流脉冲治疗可以控制mNPC的命运，并可用于开发神经系统疾病的治疗策略。该系统可用于多渠道的细胞培养、长期EF刺激、细胞形态观测和自动延时图像采集。这种微流体系统不仅缩短了所需的实验时间，而且提高了微环境控制的准确性。
Introduction
神经前体细胞（NPC，也称为神经干细胞和祖细胞）可以作为神经退行性治疗策略1的有希望的候选人。未分化的NPC具有自我更新能力、多能性和增殖能力2、3。先前的一项研究曾报道说，细胞外基质和分子介质调节NPC的分化。表皮生长因子（EGF）和基本成纤维细胞生长因子（bFGF）促进NPC增殖，从而保持无差别状态4。
先前的研究曾报道，电刺激可以调节细胞生理活动，如第5分区、迁移6、7、8、分化1、9、10和细胞死亡11。电场在中枢神经系统发育和再生中起着至关重要的作用。从2009年至2019年，该实验室调查了EF在微流体系统1、6、7、8、15、16、17中应用的细胞反应。多通道、光学透明、电触电 （MOE） 芯片设计用于免疫荧光染色，用于共焦显微镜。该芯片具有较高的光学透明度和良好的耐用性，允许在一项研究中同时进行三次独立的刺激实验和若干免疫受污染条件。微流体系统有利于简化整体实验设置，进而有利于试剂和样品的消耗。本文描述了用于长期细胞分化研究的微流体细胞培养系统的发展。
Protocol
1. MOE 芯片的设计和制造 使用适当的软件（图1A，材料表）绘制单个聚甲基甲基酸酯（PMMA）层和双面胶带的图案。用 CO2 激光机抄写机（图 1B）切割 PMMA 板材和双面胶带。 打开 CO2 激光抄写机并将其连接到个人计算机。使用软件打开设计模式文件。 将 PMMA 板（275 mm x 400 mm）或双面胶带（210 mm x 297 mm）放…
Representative Results
MOE芯片的详细配置显示在 图1中。微流体芯片为减少实验设置尺寸、样品体积和试剂体积提供了有益的方法。MOE芯片设计用于在一项研究中同时进行三个独立的EF刺激实验和几个免疫染色条件（图3）。此外，光学透明度高的MOE芯片也适合共焦显微镜检查。MOE芯片还设计用于在单个实验中同时研究不同细胞培养条件（例如，多个EF…
Discussion
在制造 MOE 芯片时，适配器将连接到 MOE 芯片的 1 层，并带有速效氰酸酯胶水。胶水涂在适配器的 4 个角上，然后均匀地施加在适配器上。必须避免胶水过量，以确保胶水完全聚合。此外，已完成的 MOE 芯片组件在真空室中孵育。此步骤有助于消除 PMMA 层、双面胶带和盖玻璃之间的气泡。
电极材料的选择是基于这样一个事实，即氯化物离子，这是丰富的存在于中等，是电解产?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢西尼卡学院生物医学研究所的唐K.唐教授在提供小鼠神经干细胞和祖细胞（mNPC）方面的帮助。作者还感谢唐克唐教授和李英山女士就跨国公司的区别化问题进行了宝贵的讨论。
Materials
| 1 mm PMMA substrates (Layers 1-3) | BHT | K2R20 | Polymethyl methacrylate (PMMA), http://www.bothharvest.com/zh-tw/product-421076/Optical-PMMA-Non-Coated-BHT-K2Rxx-xx=-thickness-choices.html |
| 15 mL plastic tube | Protech Technology Enterprise Co., Ltd | CT-15-PL-TW | Conical bottomed tube with cap, assembled, presterilized |
| 3 mL syringe | TERUMO | DVR-3413 | 3 mL oral syringes, without needle |
| 3 mm optical grade PMMA (Layer 5) | CHI MEI Corporation | ACRYPOLY PMMA Sheet | Optical grade PMMA |
| 3-way stopcock | NIPRO | NCN-3L | Sterile disposable 3-way stopcock |
| 5 mL syringe | TERUMO | DVR-3410 | 5 mL oral syringes, without needle |
| Adaptor | Dong Zhong Co., Ltd. | Customized | PMMA adaptor |
| Agarose | Sigma-Aldrich | A9414 | Agarose, low gelling temperature |
| Amplifier | A.A. Lab Systems Ltd | A-304 | High voltage amplifier |
| AutoCAD software | Autodesk | Educational Version | Drafting |
| B-27 supplement | Gibco | 12587-010 | B-27 supplement (50x), minus vitamin A |
| Basic fibroblast growth factor (bFGF) | Peprotech | AF-100-18B | Also called recombinant human FGF-basic |
| Black rubber bung | TERUMO | DVR-3413 | From 3 mL oral syringes, without needle |
| Bovine serum albumin (BSA) | Sigma-Aldrich | B4287 | Blocking reagent |
| Centrifuge | HSIANGTAI | CV2060 | Centrifuge |
| CO2 laser scriber | Laser Tools and Technics Corp. | ILS-II | Purchased from http://www.lttcorp.com/index.htm |
| Cone connector | IDEX Health & Science | F-120X | One-piece fingertight 10-32 coned, for 1/16" OD natural |
| Cone-Luer adaptor | IDEX Health & Science | P-659 | Luer Adapter 10-32 Female to Female Luer, PEEK |
| Confocal fluorescence microscope | Leica Microsystems | TCS SP5 | Leica TCS SP5 user manual, http://www3.unifr.ch/bioimage/wp-content/uploads/2013/10/User-Manual_TCS_SP5_V02_EN.pdf |
| Digital camera | OLYMPUS | E-330 | Automatic time-lapse image acquisition |
| Digital oscilloscope | Tektronix | TDS2024 | Measure voltage or current signals over time in an electronic circuit or component to display amplitude and frequency. |
| Double-sided tape | 3M | PET 8018 | Purchased from http://en.thd.com.tw/ |
| Dulbecco’s modified Eagle’s medium/Ham's nutrient mixture F-12 (DMEM/F12) | Gibco | 12400024 | DMEM/F-12, powder, HEPES |
| Dulbecco's phosphate-buffered saline (DPBS) | Gibco | 21600010 | DPBS, powder, no calcium, no magnesium |
| EF multiplexer | Asiatic Sky Co., Ltd. | Customized | Monitor and control the electric current in individual channels |
| Epidermal growth factor (EGF) | Peprotech | AF-100-15 | Also called recombinant human EGF |
| Fast-acting cyanoacrylate glue | 3M | 7004T | Strength instant adhesive (liquid) |
| Flat bottom connector | IDEX Health & Science | P-206 | Flangeless male nut Delrin, 1/4-28 flat-bottom, for 1/16" OD blue |
| Function generator | Agilent Technologies | 33120A | High-performance 15 MHz synthesized function generator with built-in arbitrary waveform capability |
| Goat anti-mouse IgG H&L (Alexa Fluor 488) | Abcam | ab150117 | Goat anti-mouse IgG H&L (Alexa Fluor 488) preadsorbed |
| Goat anti-rabbit IgG H&L (Alexa Fluor 555) | Abcam | ab150086 | Goat polyclonal secondary antibody to rabbit IgG – H&L (Alexa Fluor 555), preadsorbed |
| Hoechst 33342 | Invitrogen | H3570 | Nuclear staining |
| ImageJ software | National Institutes of Health | 1.48v | Analyze the fluorescent images |
| Indium–tin–oxide (ITO) glass | Merck | 300739 | For ITO heater |
| Inverted phase contrast microscope | OLYMPUS | CKX41 | For cell morphology observation |
| K-type thermocouple | Tecpel | TPK-02A | Temperature thermocouples |
| Luer adapter | IDEX Health & Science | P618-01 | Luer adapter female Luer to 1/4-28 male polypropylene |
| Luer lock syringe | TERUMO | DVR-3413 | For agar salt bridges |
| Mouse anti-GFAP | eBioscience | 14-9892 | Astrocytes marker |
| Oligodendrocyte marker O4 antibody | R&D Systems | MAB1326 | Oligodendrocytes marker |
| Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | Fixing agent |
| Phosphate buffered saline (PBS) | Basic Life | BL2651 | Washing solution |
| Poly-L-Lysine (PLL) | SIGMA | P4707 | Coating solution |
| Precision cover glasses thickness No. 1.5H | MARIENFELD | 107242 | https://www.marienfeld-superior.com/precision-cover-glasses-thickness-no-1-5h-tol-5-m.html |
| Programmable X-Y-Z motorised stage | Tanlian Inc | Customized | Purchased from http://www.tanlian.tw/ndex.files/motort.htm |
| Proportional–integral–derivative (PID) controller | Toho Electronics | TTM-J4-R-AB | Temperature controller |
| PTFE tube | Professional Plastics Inc. Taiwan Branch | Outer diameter 1/16 Inches | White translucent PTFE tubing |
| Rabbit anti-Tuj1 | Abcam | ab18207 | Neuron marker |
| Syringe pump | New Era Systems Inc | NE-1000 | NE-1000 programmable single syringe pump |
| TFD4 detergent | FRANKLAB | TFD4 | Cover glass cleaner |
| Thermal bonder | Kuan-MIN Tech Co. | Customized | Purchased from http://kmtco.com.tw/ |
| Triton X-100 | Sigma-Aldrich | T8787 | Permeabilized solution |
| Ultrasonic cleaner | LEO | LEO-300S | Ultrasonic steri-cleaner |
| Vacuum chamber | DENG YNG INSTRUMENTS CO., Ltd. | DOV-30 | Vacuum drying oven |
| White fingertight plug | IDEX Health & Science | P-316 | 1/4-28 Flat-Bottom, https://www.idex-hs.com/store/fluidics/fluidic-connections/plug-teflonr-pfa-1-4-28-flat-bottom.html |
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Citer Cet Article
Chang, H., Chou, S., Cheng, J. Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices. J. Vis. Exp. (170), e61917, doi:10.3791/61917 (2021).