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><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 &amp;micro;L aliquots and diluted to 20 &amp;micro;g/&amp;micro;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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

生物学

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

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 ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

微接触印刷的蛋白细胞生物学

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...