图案蛋白质和细胞的通用的方法
Summary
This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.
Abstract
Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.
Introduction
在组织工程和生物传感，控制在微米尺度的蛋白质和细胞的空间组织的能力，已成为在过去的四十年里1，2，3越来越重要。蛋白质和细胞的精确空间组织已经允许研究者检查包含相似或不同类型的细胞，以引导细胞生长，和固定生物分子的生物传感器4，5，6，7，8的制造电池和基板之间的相互作用， 9。
图案蛋白目前的方法包括光图案和微接触印刷。光图案利用其在暴露交联ULTR光敏材料紫色（UV）光。冲着光掩模（由透明区域的较深区域，以防止UV光透射）的UV光使在随后可被用于生物材料或电池10，11的后续附着特定区域交联。虽然这种方案是非常准确的，并允许培养表面的地形的精确控制，它是有限的，可以通过UV辐射12被图案化UV敏感的生物分子。微接触印刷是形成图案的特定蛋白质13,14的另一种流行的方法。在该方法中，聚二甲基硅氧烷（PDMS）印模与多种表面改性试剂中所选择的生物分子基底的溶液中浸泡前处理。它然后轻轻按压到玻璃盖玻片或其它表面从而“冲压”的生物分子上的培养物表面。何wever，冲压仅限于可以的PDMS压模15传送以及生物分子的润湿性的表面的材料的类型。
细胞的直接图案化可以更加困难，并依赖于复杂的方法，例如可切换基板，基于模版的方法，或图案形成具有特定细胞粘附分子16,17。这些方法是在自己的能力图案细胞由于缺乏兼容细胞粘附基底的限制，该方法的不相容性与敏感的生物细胞和约束，不一致性在再现图案化，并且该过程的复杂性工作。例如，与可切换的基板，定制基材需要被设计为每一个细胞类型，在暴露于切换它们遵守特定的细胞类型，而不降解到UV光和热在过程17使用的，< SUP类=“外部参照”> 18，19，20。基于模板图案的方法是在自己的能力格局细胞多才多艺的;然而，它难以制造的PDMS模具在使用16，21中的适当的厚度。细胞进入的PDMS微流体通道的直接注射有一些优点，如：1）缓解微流体通道的制造和2），用于许多不同的细胞和基质的适宜性。然而，在注射过程中空气气泡捕捉由于PDMS的不使用等离子体清洗，或其他方法来减少气泡的疏水性的普遍问题，使得难以一致地创建在玻璃或塑料表面21图案化的细胞。
这项工作扩展了毛细管微模塑22，23，小姑娘=“外部参照”> 24，25，26，并报告给注射蛋白质和细胞悬液到微通道的方法。此处所使用的方法展示衬底的图案化和特定细胞类型的直接和间接的构图。该技术克服了PDMS的疏水性高，并采取的PDMS 27的透气性的优点消除气泡的任一底物或细胞注射期间的存在。本文证明了具有几种不同的底物和细胞类型的使用的技术的。本文还使用常规的光刻法以及一种简单和低成本的粘合带的方法在资源有用有限设置28个 ，29突出的模具的制造中用于软光刻。
Protocol
Representative Results
Discussion
而常规的光刻是用于创建模具软光刻，设备，材料，和需要使用传统的光刻技术的良好建立的技术是不容易获得的大多数实验室。对于没有访问这些资源的实验室，我们已经提出了胶带制造为具有相对简单的功能，对于微流体装置产生的模具的方法。这种方法允许任何实验室创建和利用用于研究目的使用现成的工具微流体装置。粘合带的方法可以用低成本的桌面乙烯基切刀31得?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究经费是由新泽西州委员会脊髓研究（NJCSCR）（以FHK）提供，授予CSCR14IRG005（到BLF），美国国立卫生研究院授予R15NS087501（以CHC），和FM柯比基金会（以ETA）。
Materials
| CorelDRAW X4 CAD Drawing Tools | Corel Corporation, Canada | X4 Version 14.0.0.701 | CAD tool used to draw the layout of the microfluidic device |
| Laser Printer HP | Hewlett Packard, CA | 1739629 | Used to print the layout of microfluidic device for adhesive tape technique |
| Bel-Art Dessicator | Fisher Scientific, MA | 08-594-16B | Used to degass the PDMS mixture |
| Adhesive Scotch Tape | 3M Product, MN | Tape 600 | Used to fabricate adhesive tape Master |
| PDMS Sylgard 184 | Dow Corning, MI | 1064291 | Casting polymer |
| Petri Dish | Fisher Scientific, MA | 08-772-23 | Used to keep the mold to cast with PDMS |
| Stainless steel Scalpel (#3) with blade (# 11) | Feather Safety Razor Co. Ltd. Japan | 2976#11 | Used to cut the PDMS |
| Tweezers | Ted Pella, CA | 5627-07 | Used to handle the PDMS cast during peeling |
| Glass slides | Fisher Scientific, MA | 12-546-2 | Used as surface to pattern the Substrate |
| Glass slides | Fisher Scientific, MA | 12-544-4 | Used as surface to pattern the Substrate |
| Rubber Roller | Dick Blick Art Materials, IL | 40104-1004 | Used to attach adhesive tape on glass without trapping air bubbles |
| Laser Mask Writer | Heidelberg Instruments, Germany | DWL66fs | Used to fabricate quartz mask used in photolithography fabrication process |
| EVG Mask Aligner (Photolithography UV exposure tool) | EV Group, Germany | EVG 620T(B) | Used to expose the photoresist to UV light |
| Spin Coater Headway | Headway Research Inc, TX | PWM32-PS-CB15PL | Used to spin coat the photoresist on silicon wafer |
| Photoresists SU-8 50 | MicroChem, MA | Y131269 | Negative photoresist used for mold fabrication |
| SU-8 Devloper | MicroChem, MA | Y020100 | Photoresist developer |
| Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane | UCT Specialties, PA | T2492-KG | Coat mold to avoid PDMS adhesion |
| Isopropanol | Sigma-Aldrich, MO | 190764 | Cleaning Solvent |
| Ethanol | Sigma-Aldrich, MO | 24102 | Sterilization Solvent |
| Poly-D-Lysine hydrobromide (PDL) | Sigma-Aldrich, MO | P0899-10MG | PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer |
| Laminin | Sigma-Aldrich, MO | L2020 | Laminin aliquoted into 10 µL aliquots and diluted to 20 µg/µL in PBS prior to use |
| BSA | Fisher Scientific, MA | BP1605100 | Cell culture |
| C2C12 Myoblast cell lline | ATCC, VA | CRL-1722 | Used to demonstrate C2C12 patterning |
| PC12 Cell Line | ATCC, VA | CRL-1721 | Used to demonstrate PC12 patterning |
| Collagen type 1, rat tail | BD Biosciences | 40236 | Cell culture |
| DMEM | GIBCO, MA | 11965-084 | Cell culture |
| Horse Serum, heat inactivated | Fisher Scientific, MA | 26050-070 | Cell culture |
| Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) | Sigma-Aldrich, MO | P1951 | To label cells |
| Calcein-AM live dead cell Assay kit | Invitrogen, MA | L-3224 | Cell viability Assay |
| Biopsy Hole Punch | Ted Pella, CA | 15110-10 | Punched hole in PDMS |
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