设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激
Summary
Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.
Abstract
微流体设备能够创建pH值，温度，盐浓度，和其它物理或化学刺激的精确和可控的细胞微环境。他们已经通过像环境在体内提供了通常用于体外细胞研究。尤其，细胞如何响应于化学梯度，电场，应力和剪应力引起了许多利益，因为这些现象对理解细胞特性和功能非常重要。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或聚对苯二甲酸乙酯（PET）衬底。出这些材料，PMMA基底是便宜的，并且可以使用激光烧蚀和书写容易加工。虽然一些微流体装置已被设计和制造用于产生多个共存化学和电刺激，其中没有一个被认为是够在减少实验重复序列，特别是用于筛选目的有效。在这份报告中，我们描述了我们的两个基于PMMA微流控芯片设计和制造用于研究细胞反应，在生产活性氧和迁移，下单或共存化学/电气/剪切应力刺激。第一芯片产生五个相对浓度0，1/8，1/2，7/8，和1在培养的区域，与内部这些领域中产生的剪切应力梯度在一起。第二芯片产生相同的相对浓度，但与各培养区中创建的五个不同的电场强度。这些装置不仅提供细胞与精确，可控的微环境，也大大增加了实验的吞吐量。
Introduction
体内细胞通过各种生物分子，包括细胞外基质（ECM），碳水化合物，脂质，细胞和其它细胞的包围。它们官能通过响应微环境刺激如使用ECM相互作用和反应的各种生长因子的化学梯度。传统上， 在体外细胞研究是在细胞培养皿进行，其中细胞和试剂的消耗大，细胞生长在静态（非循环）环境。最近，随着流体部件集成的微制造的器件中的更可控的方式提供了用于细胞的研究的替代的平台。这样的装置能够同时最大限度地减少细胞和试剂的消耗产生的化学和物理刺激的精确微环境。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或polyethylenetere邻苯二甲酸酯（PET）的基板1-3。基于PDMS的设备是透明的，生物相容的，并能透过气体，使它们适合于长期的细胞培养和研究。 PMMA和PET基材是廉价和易于使用激光烧蚀和写作进行处理。
微流体装置应提供细胞与稳定可控的微环境，其中细胞受到不同的化学和物理刺激。例如，微流体芯片被用来研究细胞的趋化性。而不是雇用Boyden小室和毛细管4,5这些微型流体装置能产生精确的化学梯度用于研究细胞的行为1,6,7传统的方法。另一例子是研究细胞的下电场功率（EFs），一个electrotaxis现象名为定向迁移。据报道细胞Electrotactic行为可能与神经再生8，胚胎发育9和伤口愈合10,11。许多研究已经进行了调查各种细胞类型，包括癌细胞12,13的electrotaxis，淋巴细胞14,15，白血病细胞11，和干细胞16。按照惯例，培养皿和盖玻片用于构建electrotactic室产生的EF 17。这种简单的设置姿势介质蒸发和不精确EF的问题，但是它们可以通过封闭的，明确定义的流体通道12,18,19的微流体装置来克服。
为了系统地研究下精确，可控化学和电刺激细胞的反应，这将是很有用处的发展能够同时提供多个刺激细胞的微流体装置。例如，Li 等人 。报告基于PDMS-微流体装置，用于创建单个或共存化学梯度和EFS 20。 Kao 等 。开发私奔类似微流体芯片由EF文件6调节肺癌细胞的趋化性。此外，为了提高吞吐量，侯等人 。设计和制作基于PMMA-多声道的双电场芯片，提供细胞与8种不同组合的刺激，是（2 EF优势×4化学物浓度）21。为了进一步增加整个并添加剪切应力刺激，我们开发了两个PMMA基微流体装置根据单个或共存化学/电/剪切应力刺激研究细胞应答。
由Lo 等 22,23报道，这些设备包括五个独立的细胞培养渠道受到持续流体流动，模仿体内循环系统。在第一芯片（化学剪切应力芯片或CSS的芯片），五个相对浓度为0，1/8，1/2，7/8，和1在培养区域生成，并剪切应力梯度是机生产线土木工程署内各五个文化领域。在第二芯片（该化学电场芯片或CEF芯片），通过使用一个单组电极和第2注射泵中，除了这些培养区域内五个不同的化学浓度时，产生五个EF强度。数值计算和模拟执行，以便更好地设计和操作这些芯片，而这些设备内培养肺癌细胞受到单个或共存刺激用于相对于观察到的活性氧物种的它们的响应（ROS），迁移率，和运移方向。这些芯片被证明是节省时间，高吞吐量和可靠的器件用于研究细胞对各种微环境刺激的反应。
Protocol
Representative Results
Discussion
PMMA基芯片使用激光烧蚀和写入与基于PDMS芯片，从而需要更复杂的软光刻当它们是便宜和更容易的方法制造。设计的微流体芯片之后，在制造和装配可以只在5分钟内完成。有迹象表明，应注意在执行实验中支付给一些关键的步骤。首先是“组装”的问题。适配器应适当粘在最上面的层的芯片。如果施加太大胶可能泄漏进入流体通道，并且如果使用太少的胶液可能泄漏出来的芯片。另外，在组装的…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.
Materials
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11965-092 | Cell culture medium |
| Trypsin | Gibco | 25300-054 | detach cell from the dish |
| Fetal bovine serum (FBS) | Gibco | 10082147 | Cell culture medium |
| 10-cm cell culture Petri dish | Nunc | 150350 | Cell culture |
| Bright-Line Hemacytometer | Sigma | Z359629 | Cell Counting Equipment |
| PMMA | Customized | Customized | Microfluidic chip |
| Adaptor | Customized | Customized | Microfluidic chip |
| 0.07/0.22 mm double-sided tape | 3M | 8018/9088 | Microfluidic chip |
| Low melting point agarose | Sigma | A9414 | Salt bridge |
| 2'-7'-dichlorodihydrofluoresce diacetate | Sigma | D6883 | Intracellular ROS measurement |
| Indium tin oxide (ITO) glass | Merck | 300739 | Heater |
| Proportional-integral-derivative controller | JETEC Electronics Co. | TTM-J4-R-AB | Temperature controller |
| Thermal coupler | TECPEL | TPK-02A | Temperature controller |
| CO2 laser scriber | Laser Tools & Technics Corp. | ILS2 | Microfluidic chip fabrication |
| Syringe pumps | New Era | NE-300 | Pumping medium and chemicals into the chip |
| Power supply | Major Science | MP-300V | Supplying direct currents |
| Inverted microscope | Olympus | CKX41 | Monitoring cell migration |
| Inverted fluorescent microscope | Nikon | TS-100 | Monitoring cell migartion and fluorescencent signals |
| DSLR camera | Canon | 60D | Recording bright-field images |
| CCD camera | Nikon | DS-Qi1 | Recording fluorescent images |
| super glue | 3M Scotch | 7004 | Attaching adaptors to PMMA substrates |
| AutoCAD | Autodesk Inc. | Designing microfluidic chips | |
| DMSO | Sigma | D8418 | Dissolving DCFDA |
| ImgeJ | National Institutes of Health | Quantifying fluorescent intensities and cell migration |
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