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）。

注意:使用前请咨询所有相关的材料安全数据表（MSDS）。一些在该协议中使用的化学品是有毒和致癌性。使用有毒或酸/碱材料时，请使用所有适当安全操作规范（通风橱，手套箱）和个人防护装备（护目镜，手套，实验室外套，全长裤，封闭趾鞋）。 1.制造主模具软光刻使用光刻<ol><li>绘制使用计算机辅助设计（CAD）绘图工具的微通道的布局。 </li><li>使用激光掩膜写入空白掩模板打印的布局。 </li><li>冲洗用丙酮随后异丙醇和干燥用氮气气枪一个4英寸的硅晶片，以确保没有溶剂残留在晶片上。 </li><li>通过在200℃下烘烤的热板上进行5分钟脱水晶片。 </li><li>选择设计的微通道的所需高度的负光致抗蚀剂。 F或者例如，使用负光致抗蚀剂的SU-8 50具有50μm的高度，以获得微通道。 </li><li>允许晶片冷却至室温，然后对齐晶片上旋涂器的卡盘。 </li><li>分配负光致抗蚀剂4毫升（毫升/英寸直径晶片的1）到晶片的中心。 </li><li>旋涂在晶片用旋涂器的两步旋转循环。首先，应用500rpm的传播周期以100rpm / s的的10秒的加速度。然后应用2000 rpm的旋转周期为300转/秒2的加速度为30秒，以获得在晶片上的光刻胶的一个50微米的涂层。 </li><li>软烘烤在根据制造商的指示在热板上两个步骤的晶片。对于光致抗蚀剂50μm的涂层，第一预烘烤在65℃下进行6分钟的晶片，然后立即斜坡上升的热板温度进行后烘烤在95℃下将晶片20分钟。 </li><li>允许晶片冷却到室温，然后装入晶圆上光刻阶段。 </li><li>使用掩模对准对准晶片上的掩模，并在为146秒的1.5毫瓦/厘米2的强度曝光晶片于UV光（根据制造商的说明）应用220毫焦耳的总UV剂量/ cm 2的所需一50微米厚的一层光刻胶。 </li><li>在热板上曝光后烘烤应用于晶片在两个步骤。对于光致抗蚀剂的一个50微米的涂层，第一预烘烤在65℃下将晶片1分钟，并立即斜坡上升的热板温度进行后烘焙它在95℃下5分钟。 </li><li>在SU-8开发者淹没晶圆并轻轻晃动，直到特点成为晶圆上明确的发展晶圆英寸制定为50微米厚的光刻胶〜6分钟的片。 </li><li>冲洗掉多余的开发商用异丙醇和乙醇，然后用干燥氮气气枪晶圆。 </li><li>硬烤在硅晶片上的热板上在150℃下进行15分钟。 </li><li>放置晶片与硅烷化剂的25微升干燥器十三氟-1,1,2,2-四氢辛基-1-三氯硅烷。 注:硅烷化剂具有腐蚀性，因此应在酸/碱通风橱中使用，配备适当的个人防护设备（防护眼镜，手套，实验室外套，全长裤，封闭趾鞋）。 </li><li>施加真空5分钟，并暴露所述晶片的硅烷30分钟的蒸汽而不释放真空。 </li><li>转移晶片到适当大小的培养皿。 </li></ol> 2.制造主模具软光刻使用胶带<ol><li>绘制使用CAD绘图工具的微通道的布局，并打印在白纸上的绘图。 </li><li>清洁载玻片大到足以容纳用异丙醇设计，然后干燥用空气枪的幻灯片。 </li><li>安装胶带到清洁玻片。要小心，不要捕获任何气泡。 </li><li>大盘的GLA侧面SS拨动到纸上的设计，与磁带的一面朝上。 </li><li>使用手术刀使用的纸张设计作为参考，以削减在载玻片磁带，然后从载玻片上的不需要的区域剥离胶带。 </li><li>冲洗用异丙醇载玻片，然后干燥用空气枪。烘烤在烘箱载玻片在65℃下30分钟。 </li><li>在磁带轻轻摇动用橡胶辊以除去气泡并允许滑动冷却到室温。 </li></ol> 3. PDMS设备的软光刻制造<ol><li>混合的PDMS弹性体及其固化剂以10:1的比例（重量:重量），充分搅拌该混合物剧烈，然后将其放入一个真空室脱气混合物直至没有气泡出现在混合物中。 </li><li>倒入混合物到硅烷化硅模具或胶带铸型在培养皿以获得〜2毫米的PDMS厚层和脱气，直至所有的气泡消失。 </li><li>固化的PDMS在烘箱在65℃下2小时。 </li><li>用手术刀切割PDMS层从功能至少5mm远，然后剥离使用镊子模具中的固化的PDMS。 </li><li>冲与1毫米活检单个入口孔的任何地方打孔的微通道，优选在微通道的一个网络的端部如在图2a可见和4b。 </li><li>请用胶带蒙上除去吸附到设备上的灰尘颗粒的PDMS。通过用70％乙醇，随后通过无菌去离子（DI）水的溶液冲洗消毒的PDMS微通道装置（PDMS铸），并暴露于紫外线30分钟。 </li><li>保持无菌PDMS设备在无菌培养皿直至使用。 </li></ol> 4.基材图案<ol><li>放置用镊子投无菌的PDMS上的无菌玻璃盖玻片或培养皿并使用镊子的顶端施加轻柔的压力。 注:PDMS投使得保形，但与GL可逆密封屁股盖玻片或培养皿中不使用任何粘合剂形成微通道。 </li><li>放置一个微滴 - 上完全覆盖入口孔的入口处的底物溶液（20 40微升）。图案聚-D-赖氨酸（PDL）通过在微通道的入口放置的PDL的20微升液滴在四硼酸钠缓冲液中。 </li><li>把培养皿在〜254 mmHgA的真空（相当于容纳在生物实验室真空）10分钟，并观察空气流出通道中的气泡的形式。 </li><li>释放真空并观察流入微通道的底物溶液。 </li><li>在孵育底物附着在适当的条件下菜。 见表1和表 2的培养条件（这些取决于在基片上）。对于PDL图案，在37℃孵育1小时。 </li><li>用无菌镊子小心地剥去PDMS邮票，用去离子水洗涤三次对玻璃盖玻片的格局。 </li&gt; <li> 1％的牛血清白蛋白（BSA）溶液添加到培养皿，以覆盖盘或玻璃盖玻片并孵育过夜，在37℃。 注意:这将减少在未涂覆区域的非特异性粘附。 </li><li>吸掉1％BSA的溶液中，将图案化衬底，现在可以使用了。 </li></ol> 5.细胞间接图案<ol><li>制备在无血清培养基中的细胞悬浮液。细胞类型和细胞密度都列于表1。 </li><li>将细胞悬浮液添加到该图案化的培养皿和完全淹没在细胞悬浮液中的图案化区域。 </li><li>孵育在37℃的培养皿中15分钟的培养箱，以允许细胞附着到图案化衬底。 </li><li>吸从培养皿多余的细胞悬浮液，并用磷酸盐缓冲盐水（PBS）洗涤所述图案化的细胞三次，同时轻轻10秒摇动以除去未附着的细胞。 </li><li>添加一个ppropriate培养基到图案化的细胞培养物，并在CO 2培养箱中孵育该图案化细胞在37℃。 </li></ol> 6.细胞直接图案注意:此技术是在步骤5。然而，在步骤5中描述的，不同的是间接细胞图案的替代，在该技术的细胞上具有或不具有底物涂层的组织培养表面图案化。 <ol><li>通过用乙醇接着DI水中70％的溶液冲洗消毒微流体装置。 </li><li>过夜浸泡装置在1％BSA的溶液中，以防止细胞粘附到PDMS。 </li><li>干燥设备在室温和其附加到组织培养处理的培养皿底部。 </li><li>制备在无血清培养基中的细胞悬浮液。细胞类型和细胞密度都列于表2中 。 </li><li>放置一个微滴 - 细胞悬浮液（4 8微升），足以填满微通道，在入口完全覆盖入口孔。 </li><li>把培养皿在真空10分钟，并观察空气流出通道中的气泡的形式。释放真空并观察流入微通道中的细胞悬浮液。 </li><li>孵化培养皿在培养箱，以促进细胞粘附根据培养条件（取决于细胞类型图案化的）在表2中提及。添加PBS的培养皿浸没PDMS设备。 </li><li>皮尔的PDMS小心掉培养皿中用镊子和用PBS洗模式。细胞培养基添加到培养皿，然后将细胞培养物在孵化器。 </li></ol>

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B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chips+&amp;+tips:+simple+fabrication+of+microfluidic+devices+by+replicating+scotch-tape+masters.">Chips &amp; tips: simple fabrication of microfluidic devices by replicating scotch-tape masters.</a> Lab Chip. , (2010).</li><li>Anil, B. S., Frank, H. K., Derek, Y., Cheul, H. C., Ellen, T. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuum-assisted+fluid+flow+in+microchannels+to+pattern+substrates+and+cells.">Vacuum-assisted fluid flow in microchannels to pattern substrates and cells.</a> Biofabrication. 6 (3), 035016 (2014).</li><li>Yuen, P. K., Goral, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+rapid+prototyping+of+flexible+microfluidic+devices+using+a+desktop+digital+craft+cutter.">Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter.</a> Lab on a Chip. 10 (3), 384-387 (2010).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-loading+and+cell+culture+in+one+layer+microfluidic+devices.">Self-loading and cell culture in one layer microfluidic devices.</a> Biomed Microdevices. 11 (3), 679-684 (2009).</li><li>Feng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mammalian+cells+under+high+vacuum+condition+for+ion+bombardment.">Survival of mammalian cells under high vacuum condition for ion bombardment.</a> Cryobiology. 49 (3), 241-249 (2004).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab on a Chip. 6 (12), 1548-1549 (2006).</li><li>Fan, D. -. H., Yuan, S. -. W., Shen, Y. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+with+BSA+blocking+based+on+in+situ+synthesized+gold+nanoparticles+in+poly+(dimethylsiloxane)+microchip.">Surface modification with BSA blocking based on in situ synthesized gold nanoparticles in poly (dimethylsiloxane) microchip.</a> Colloids Surf, B. 75 (2), 608-611 (2010).</li><li>Hideshima, S., Sato, R., Inoue, S., Kuroiwa, S., Osaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+tumor+marker+in+blood+serum+using+antibody-modified+field+effect+transistor+with+optimized+BSA+blocking.">Detection of tumor marker in blood serum using antibody-modified field effect transistor with optimized BSA blocking.</a> Sens Actuator B-Chem. 161 (1), 146-150 (2012).</li><li>Zheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+immunoassay+through+in-channel+microfluidic+patterning.">High-throughput immunoassay through in-channel microfluidic patterning.</a> Lab on a Chip. 12 (14), 2487-2490 (2012).</li><li>MacLeish, P., Barnstable, C., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Procs Nat Acad of Sci. 80 (22), 7014-7018 (1983).</li><li>Suchodolskis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+chemically+modified+baker's+yeast+cells+studied+by+AFM.">Elastic properties of chemically modified baker's yeast cells studied by AFM.</a> Surf Interface Anal. 43 (13), 1636-1640 (2011).</li></ol>

The authors declare no competing financial interests.

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?...

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，&lt;<html> SUP类="外部参照"&gt; 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"&gt; 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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CorelDRAW X4 CAD Drawing Tools</td>
			<td>Corel Corporation, Canada</td>
			<td>X4 Version 14.0.0.701</td>
			<td>CAD tool used to draw the layout of the microfluidic device</td>
		</tr>
		<tr>
			<td>Laser Printer HP</td>
			<td>Hewlett Packard, CA</td>
			<td>1739629</td>
			<td>Used to print the layout of microfluidic device for adhesive tape technique</td>
		</tr>
		<tr>
			<td>Bel-Art Dessicator</td>
			<td>Fisher Scientific, MA</td>
			<td>08-594-16B</td>
			<td>Used to degass the PDMS mixture</td>
		</tr>
		<tr>
			<td>Adhesive Scotch Tape</td>
			<td>3M Product, MN</td>
			<td>Tape 600</td>
			<td>Used to fabricate adhesive tape Master</td>
		</tr>
		<tr>
			<td>PDMS Sylgard 184</td>
			<td>Dow Corning, MI</td>
			<td>1064291</td>
			<td>Casting polymer</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific, MA</td>
			<td>08-772-23</td>
			<td>Used to keep the mold to cast with PDMS</td>
		</tr>
		<tr>
			<td>Stainless steel Scalpel (#3) with blade (# 11)</td>
			<td>Feather Safety Razor Co. Ltd. Japan</td>
			<td>2976#11</td>
			<td>Used to cut the PDMS</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ted Pella, CA</td>
			<td>5627-07</td>
			<td>Used to handle the PDMS cast during peeling</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific, MA</td>
			<td>12-546-2</td>
			<td>Used as surface to pattern the Substrate</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific, MA</td>
			<td>12-544-4</td>
			<td>Used as surface to pattern the Substrate</td>
		</tr>
		<tr>
			<td>Rubber Roller</td>
			<td>Dick Blick Art Materials, IL</td>
			<td>40104-1004</td>
			<td>Used to attach adhesive tape on glass without trapping air bubbles</td>
		</tr>
		<tr>
			<td>Laser Mask Writer</td>
			<td>Heidelberg Instruments, Germany</td>
			<td>DWL66fs</td>
			<td>Used to fabricate quartz mask used in photolithography fabrication process</td>
		</tr>
		<tr>
			<td>EVG Mask Aligner (Photolithography UV exposure tool)</td>
			<td>EV Group, Germany</td>
			<td>EVG 620T(B)</td>
			<td>Used to expose the photoresist to UV light</td>
		</tr>
		<tr>
			<td>Spin Coater Headway</td>
			<td>Headway Research Inc, TX</td>
			<td>PWM32-PS-CB15PL</td>
			<td>Used to spin coat the photoresist on silicon wafer</td>
		</tr>
		<tr>
			<td>Photoresists SU-8 50</td>
			<td>MicroChem, MA</td>
			<td>Y131269</td>
			<td>Negative photoresist used for mold fabrication</td>
		</tr>
		<tr>
			<td>SU-8 Devloper</td>
			<td>MicroChem, MA</td>
			<td>Y020100</td>
			<td>Photoresist developer</td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane</td>
			<td>UCT Specialties, PA</td>
			<td>T2492-KG</td>
			<td>Coat mold to avoid PDMS adhesion</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, MO</td>
			<td>190764</td>
			<td>Cleaning Solvent</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, MO</td>
			<td>24102</td>
			<td>Sterilization Solvent</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine hydrobromide (PDL)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>P0899-10MG</td>
			<td>PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich, MO</td>
			<td>L2020</td>
			<td>Laminin aliquoted into 10 &#181;L aliquots and diluted to 20 &#181;g/&#181;L in PBS prior to use</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific, MA</td>
			<td>BP1605100</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>C2C12 Myoblast cell lline</td>
			<td>ATCC, VA</td>
			<td>CRL-1722</td>
			<td>Used to demonstrate C2C12 patterning</td>
		</tr>
		<tr>
			<td>PC12 Cell Line</td>
			<td>ATCC, VA</td>
			<td>CRL-1721</td>
			<td>Used to demonstrate PC12 patterning</td>
		</tr>
		<tr>
			<td>Collagen type 1, rat tail</td>
			<td>BD Biosciences</td>
			<td>40236</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO, MA</td>
			<td>11965-084</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated</td>
			<td>Fisher Scientific, MA</td>
			<td>26050-070</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>P1951</td>
			<td>To label cells</td>
		</tr>
		<tr>
			<td>Calcein-AM live dead cell Assay kit</td>
			<td>Invitrogen, MA</td>
			<td>L-3224</td>
			<td>Cell viability Assay</td>
		</tr>
		<tr>
			<td>Biopsy Hole Punch</td>
			<td>Ted Pella, CA</td>
			<td>15110-10</td>
			<td>Punched hole in PDMS</td>
		</tr>
	</tbody>
</table>

a versatile method of patterning proteins and cells

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.

此方法允许蛋白质和使用具有尺寸小至10微米的和可用的设备中几乎所有的生物实验室死胡同微流体通道，一旦母模是由细胞的间接形成图案的图案形成。这种技术可以使用传统的软光刻法，或用胶带的制造（ 图1）28，29中创建的PDMS微流体通道创建的PDMS微流体通道被利用。之前我们已经证实在神经元细胞在光感受?...

Watch this Scientific Journal Video about A Versatile Method of Patterning Proteins and Cells at JoVE.com

A Versatile Method of Patterning Proteins and Cells

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

a-versatile-method-of-patterning-proteins-and-cells

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Rutgers University. This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

视频: A Versatile Method of Patterning Proteins and Cells

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案:二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...