使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究
Summary
Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.
Abstract
定向细胞迁移的下一个直流电场（dcEF）的行为被称为electrotaxis。生理dcEF在胚胎发育，细胞分化和伤口愈合过程中引导细胞运动的显著的作用已被证明在许多研究。通过将微流体芯片的electrotaxis测定中，调查过程缩短和实验误差最小化。近年来，微流体装置制成的聚合物质（例如，甲基丙烯酸甲酯，聚甲基丙烯酸甲酯，或丙烯酸）或聚二甲基硅氧烷（PDMS）已被广泛用于研究细胞的响应电刺激。但是，与制造一个PDMS装置所需的许多步骤中，丙烯酸类微FL uidic芯片的简单而快速的结构使得它适合于设备的原型和生产。然而，没有报道的设备，这有助于该组分化学DCE的效率研究对细胞˚F影响。在这份报告中，我们描述我们的设计和丙烯酸系多信道双电场（MDF）芯片调查化学和电刺激对肺癌细胞的效果并发的制造。中密度纤维板芯片提供了一个测试的电气/化学刺激八种组合。该芯片不仅大大缩短了实验所需的时间，但在electrotaxis研究也提高了准确性。
Introduction
贴壁细胞下的直流电场（dcEF）朝向阳极或阴极移动的行为被称为electrotaxis。细胞的行为electrotactic起着胚胎发生，神经再生，和伤口愈合一个显著作用。1肿瘤细胞如鼠前列腺癌细胞，2-乳腺癌细胞，3和肺腺癌细胞4-8已根据所施加的dcEF所示electrotactic运动。生理EF已在腺组织进行了测量。9,10- Electrotaxis也有报道在腺相关的肿瘤细胞。2,3两者合计，癌细胞的electrotaxis被认为是一个转移因子11控制的电指导dcEF下的癌细胞可以是用于癌症的未来治疗的潜在的方法。然而，今天，electrotaxis的具体分子机制仍存在争议。因此，INF进行调查对癌症细胞迁移的电刺激的luence可以方便的策略的发展为癌症的治疗。
最近，生物微流体装置已被制造用于研究的细胞响应流动的剪切力，12化学梯度，13和电刺激4 体外 。使用聚二甲基硅氧烷（PDMS）或聚甲基丙烯酸甲酯（PMMA，也被称为丙烯酸类）的生物微流体装置的制造已成功地减少了此类实验的故障率。此外，使用基于丙烯酸的微流体装置作为原型调查生物受试者比使用PDMS芯片简单。在丙烯酸类装置的各种功能已经开发了用于electrotaxis研究。然而，没有任何先前的设计都能够同时测试的各种化学条件对细胞的影响，并在电场为electrotaxis研究。因此，我们开发了一种微流体装置，所述亩ltichannel双电场（MDF）四个独立培养通道和八个不同的实验条件在一个芯片包含芯片。
丙烯酸基密度板芯片，首先报道了侯等人，8集成电刺激和几个化学隔离通道。这些化学隔离通道可以用来培养在一个实验中不同类型的细胞。所述dcEF在通道是由一电源产生的。两个独立的电场，一用施加的电场强度（EFS）和另一用0 EFS，在各化学分离的信道进行的。以这种方式，在芯片提供了更好的控制的共存EF和化学刺激。此外，从MDF芯片内部的化学扩散数值模拟结果表明，无交叉污染，24小时的试验期后，通道之间发生。8
相比于叠维策报道李等人，14密度板芯片提供了一个更大的文化区域，其允许电刺激的细胞的进一步的生化分析。此外，与中密度纤维板芯片的较大的观察区域，更多的细胞可以在测试中观察，所以迁移速度的电刺激的细胞的或指向性的分析更准确。报告由Huang等人4和Tsai等人。15以前的研究的单通道芯片设计允许被测试仅一种类型的细胞或化学的。然而，中密度纤维板芯片可用于研究的各种化学品上electrotaxis的影响，以及电刺激对不同类型的细胞的作用。换句话说，中密度纤维板芯片允许对化学剂量依赖性的高效学习。
Protocol
Representative Results
Discussion
我们发现秉承丙烯酸适配器到MDF芯片的1层是棘手的过程。仅1至2微升的强力胶的应用是足够坚定奉行适配器到MDF芯片。更大量的胶水导致强力胶和不遵守的一个不完整的聚合。一旦丙烯酸适配器牢固地附着在芯片中密度纤维板，液体泄漏的微流体系统很少发生。此外，O / N培养在真空室内有助于去除双面胶带/盖玻璃或双面胶带/丙烯酸片接口之间截留空气。因此，该过程增强了培养室中密度板芯片…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.
Materials
| Reagent | |||
| DMEM medium | Gibco,Invitrogen, USA | 12800-017 | |
| Fetal Bovine Serum | Gibco,Invitrogen, USA | 16000-044 | |
| Trypsin | Gibco,Invitrogen, USA | 25200-072 | |
| PBS | Basic Life | BL2651 | |
| Y-27632 (hydrochloride) | Cayman Chemical Co | 10005583 | |
| agarose | LONZO, USA | SeaKem LE AGAROSE | |
| syringe | Terumo | 3 ml with Luer taper | |
| 3-way stopcock | Nipro | with Luer taper | |
| PMMA (acrylic) | HiShiRon Industries CO., Ltd, Taiwan | thickness 1mm, 2mm | |
| acrylic adaptor | KuanMin Technology Co., Ltd, Taichung, Taiwan | 1/4-28 port, 10x10x6 mm | customized |
| nut | Thermo Fisher Scientific Inc. | UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x | |
| Microscope cover glass | Deckgläser, Germany | 24×60 mm | |
| double-sided tape | 3M | PET 8018 | |
| super glue | 3M | Scotch Liquid Plus Super Glue | |
| Teflon tube | HENG YI ENTERPRISE CO., LTD., Taiwan | UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING | outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scientific |
| TFD4 detergent | Franklab, France | TFD4 | |
| ultrasonic steri cleaner | LEO ULTRASONIC CO., LTD., Taiwan | ||
| Thermo bonder | KuanMin Technology Co., Ltd, Taichung, Taiwan | customized | |
| CO2 laser scriber | LTT group, Taiwan | ISL-II | |
| indium tin oxide glass (ITO glass) | AimCore Technology Co., Ltd | TN/STN, ≦10Ω | |
| proportional-integral-derivative (PID) controller | JETEC Electronics Co., Japen | TTM-J40-R-AB, | |
| K-type thermocouple | TECPEL | TPK-02A | |
| 4-channel syringe pump | KdScientific, USA | 250P | |
| DC power supply | GWInstek, Taiwan | ||
| X-Y-Z motor stage | TanLian, E-O Co. Ltd., Taiwan | customized | |
| inverted microscope | Olympus, Japan | CKX41 | |
| digital SLR camera | Canon, Japan | 60D |
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