这项工作是由威康信托临床博士研究生奖学金（ECAT）的支持。

1。制作在SiO 2的Parylene模式的：处理流程（见图1） <ol><li>使用布局编辑软件，能够读/写到岸价（加州理工学院的中级形态）或GDS-II（图形数据库系统-II）的文件设计所需的聚对二甲苯-C的配置。 CIF和GDS-II是行业标准文件格式为集成电路艺术品布局。 </li><li>委员会光罩制造到适当的微电子设备，或使内部设施是否存在。 </li><li>在950℃下氧化的硅晶片在常压卧式炉（H 2 1.88 SLM和O 2 1.25 SLM）40分钟，以产生一200 nm的SiO 2层（厚度确认用小点的光谱反射率计）。 </li><li>素的氧化晶片用硅烷粘合促进剂。现在沉积的聚对二甲苯-C在22℃时的二聚物1.298纳米/毫克使用专门设计以沉积聚对二甲苯真空沉积系统的速度。 A 100nm厚的聚对二甲苯-C涂层用于如下所示的所有例子。 </li><li>下一个存款六甲基二硅氮烷（HMDS），粘合促进剂使用合适的光致抗蚀剂涂层系统中的聚对二甲苯涂覆的晶片上。 </li><li>现在旋转晶片以4,000 rpm离心30秒，而施加正光刻胶（导致1μm的理论厚度），使用相同的光致抗蚀剂涂层系统如上述。 </li><li>在90℃下软烘烤该晶片60秒</li><li>插入两个晶片和预制造光掩模成掩模对准器。 </li><li>暴露的光致抗蚀剂涂覆的晶片用所需的聚对二甲苯-C结构的紫外负表示。 </li><li>在110℃下烘烤曝光的晶片60秒</li><li>删除所有暴露的晶圆在适当的显影液显影光致抗蚀剂。 </li><li>蚀刻掉未受保护的聚对二甲苯。使用氧等离子体刻蚀系统（在50毫托腔室压力，49 sccm的O 2，100瓦的RF功率13.56MHz，并且为100nm /分钟）的蚀刻速率，以露出底下的SiO 2。 </li><li>使用适当的切割锯（主轴转速30,000 rpm，填料速度7毫米/秒）切块的晶圆。 </li><li>芯片冲洗用去离子H 2 O和氮气吹干。 </li><li>存储芯片（无限期）在无尘箱，直到需要。 </li></ol> 2。芯片的清洗和激活：协议<ol><li>洗丙酮，持续10秒从芯片去除残留的光刻胶。 </li><li>在冲洗用去离子H 2 O的3倍。 </li><li>弥补新鲜食人鱼酸（5:3比例的30％过氧化氢和98％硫酸）。 注意：准备食人鱼酸小心翼翼。它是一个非常强大的氧化剂，是强酸性，并混合过氧化氢与硫酸反应是放热反应。在酸性通风柜执行这个阶段。允许2分钟通过混合食人鱼酸，但后20分钟内使用。 </li><li>清洁和蚀刻芯片浸入PIranha酸10分钟。 </li><li>冲洗3倍筹码在去离子H 2 O和转移到无菌培养皿中。 </li><li>现在激活芯片细胞图案。例如，添加每孔两个芯片到6孔板，然后加入2ml胎牛血清，充分沉浸在所有的筹码。执行此操作，和所有随后的细胞培养阶段，在层流组织培养罩在无菌条件下。 </li><li>在孵育血清3-12小时的芯片在37°C 注：步骤2.4-2.7应按顺序和无级之间的延迟执行。如果血清激活由≥24小时后延迟食人鱼处理，细胞图案是由于来自SiO 2区减少细胞排斥减值。 可以代替胎牛血清的可以使用含有特定细胞粘附蛋白替代激活解决方案。这样的解决方案改变的两种截然不同的底物（见代表性的结果的例子）的细胞粘附特性。 </li&gt; </ol> 3。电镀细胞系片：协议<ol><li>从它们的活化溶液中除去芯片和洗一次，持续​​10秒的Hank平衡盐溶液。 </li><li>芯片放置在一个良好的文化和板选择的细胞类型作为其一贯的生长介质的悬浮液。最佳细胞铺板密度既取决于细胞类型和片上的聚对二甲苯-C的几何图案。 5×10 4个细胞/ ml的密度是一个明智的起点。 </li><li>成像是针对底层的动机细胞图案，但活细胞的行为可以很容易地使用解剖显微镜和数码相机用合适的中继透镜评估。 </li></ol>

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作者宣称没有竞争的财务权益。

芯片在食人鱼酸浸不仅用于除去任何残余的有机材料，但也蚀刻在衬底表面上。这是关键，为了能够有效地激活胎牛血清。如果不这样做可防止细胞图案化和深刻地改变芯片上细胞的行为。有没有要求清洗与食人鱼酸后消毒芯片。确实杀菌的紫外线照射已显示出削弱以剂量依赖的方式13细胞图案。必须注意的光刻工艺后洗掉所有的残余光致抗蚀剂。坚持光刻胶可以充当覆盖图案由parylene-C/SiO 2的几何形状决定的不必要的细胞质粘合层。使用上面，并与规定的试剂中所述的光刻工艺时，丙酮是有效的。然而，其他类型的光致抗蚀剂可能需要不同的溶剂中进行。 评估differe的影响，并成功nt的制造步骤中，有两种截然不同的基板的接触角可以被测量。 图2示出在芯片激活过程中发生的改变。这是有可能的，但是，在特定的血清粘合剂和排斥力蛋白质组分最终使聚对二甲苯图案芯片以发挥其各自的细胞质粘性或细胞质推斥特性。 所有代表性的成果使用的芯片为100 nm的聚对二甲苯厚度，尽管我们已经同时使用较厚和较薄的聚对二甲苯层成功地图案。重要的是，这种光刻技术允许的聚对二甲苯配置更大的三维控制比这里的说明。例如，在使用光掩模的组合，它可以创建混合厚度的聚对二甲苯的区域。这将打开的方式与定义的三维地形生成的细胞培养，超越的细胞粘附/斥力简单地发号施令地区锡永，潜在地提供集成的微流体通道到所述构建的一种手段。 如图所示，但是，该图案化平台不是普遍横跨细胞类型有效。不同的细胞系，与他们不同的细胞粘附分子型材，勿庸置疑有不同的行为在这个平台上培养时。我们还没有发现血清中的关键部件，也没有免费的细胞膜受体，它巩固这种细胞图案化平台。在未来这样做的承诺，以扩大其实用性和特异性。例如，一个“非图案化”的细胞系可被遗传修饰，以表达所需的粘附分子，因此促进形成图案。

可以理解，听写细胞粘附和形成图案的合成材料的机制是用于诸如组织工程，药物发现和生物传感器1-3的制造中很重要的。许多技术可用，演变，影响细胞粘附的无数生物，化学和物理因素的每次服用的优势。 在这里，我们描述了一个，利用微电子制造的目的最初开发过程中的细胞图案化技术。因此，该平台是占尽天时地利，使微电子技术，如多边环境协定下游一体化，进入图形化平台。 细胞膜和相邻材料之间的界面是双向的和复杂的。 在体内 ，胞外基质蛋白提供结构和强度，并通过与细胞粘附受体的相互作用在细胞行为的影响。同样，细胞v中ITRO与合成底物通过蛋白4的吸收的层相互作用而物理-化学影响也调节粘合力。例如，聚合物表面可以呈现更多的“润湿”（亲水的）通过离子或紫外线照射，或蚀刻用酸或氢氧化5治疗。用于细胞图案建立的方法利用这些和其他细胞粘附介质。例子包括喷墨印刷6，微接触压印7，物理固定8，微流体9，实时操作10，和选择性的分子组装形成图案（SMAP）11。每个人都有特定的优点和局限性。在我们工作的一个关键驱动因素，但是，是细胞图案集成微机电系统（MEMS）。 微机电系统是指由电力驱动的非常小的机械装置。这与纳米相当于nanoelectromech重叠anical系统。这个概念变成了现实，只有当半导体的策略使制造要发生在微尺度。半导体电子独自开发的微​​细加工技术已在不经意间被发现的其他用途，如细胞电很有用，例如。一个关键的下游目标是这样的微电子技术，具有高的保真度细胞图案化工艺（形成微机电装置）结合。几个现有的及以其他方式可靠，实用的细胞图案化技术是这种想法不兼容。例如，任意的嵌入式微电子学或生物传感器的精确对齐是至关重要的它们的功效，但极难实现使用技术如微接触压印。 为了解决这个问题，我们正在使用光刻印刷聚对二甲苯-C的SiO 2为基础的图形平台。光刻涉及的几何特征从转让掩模，通过紫外线照射的基片。蒙版是使用适当的计算机辅助设计程序设计。后的玻璃板上，薄薄的一层不透明的铬的代表所需的几何图案（1-2毫米的特征分辨率是可能的）。要形成图案的基板上涂有一薄层光阻剂（紫外线敏感的聚合物）。涂覆的聚合物然后对准并使其与掩模紧密接触。可以用紫外线源被应用，使得未受保护的区域进行照射，因此变得可溶并可除去在接下来的显影步骤，使掩模图案的聚对亚二甲苯-C表示后面。这个过程起源的半导体器件的开发期间。因此，硅晶片被频繁用作基板。的聚对二甲苯-C在SiO 2光刻沉积，因此是一个简单而可靠的过程，通常发生在微电子洁净室设施。 虽然聚对二甲苯有几个理想的生物工程特性（化学惰性，非生物降解的），一个限制其在细胞图案直接使用的因素是其天生的穷细胞的粘附性，部分归因于它的极端疏水性。然而，聚对二甲苯-C先前已经间接地用于细胞图案化，例如作为可剥离的蜂窝模板12,13。这种方法是通过分辨率差的限制，需要多个步骤。这里所描述的过程，而不是利用酸蚀刻步骤，随后血清孵育，以确保聚对二甲苯-C的区域成为细胞粘接性，通过降低疏水性和血清蛋白结合的结合。 最终的结果是一个两个不同基材其中，生物活化后，表现出各自的细胞质粘性或细胞质-斥力等特性代表一个有效的细胞图案化平台组成的构建体。重要的是，没有必要引入生物股份公司ENTS进入无尘室设施作为图案化衬底可被存储为使用无限期之前（于是他们正在使用的胎牛血清或另一个激活溶液激活）。 因此，本parylene-C/SiO 2图案平台是一个很好的候选人与MEMS元件的联盟，因为制造工艺如此密切反映那些用于微电子制造。

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			<td height="21" style="height:21px;width:383px;">Layout editor software package&#160;</td>
			<td style="width:572px;">e.g. CleWin 5.0 from WieWeb, http://www.wieweb.com/ns6/index.html</td>
			<td style="width:215px;"></td>
			<td style="width:615px;">Capable of reading/writing CIF or GDS-II files. Used to create parylene design for photo mask manufacture</td>
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			<td height="21" style="height:21px;width:383px;">Bespoke photo mask</td>
			<td colspan="2">e.g. Compugraphics International Ltd, Glenrothes, Scotland, www.compugraphics-photomasks.com</td>
			<td>Either fabricate in-house of facilities exist or commision</td>
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			<td height="21" style="height:21px;width:383px;">3&#34; Silicon wafers</td>
			<td>e.g. Siltronix, Archamps, France, http://www.siltronix.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atmospheric horizontal furnace</td>
			<td style="width:572px;">e.g. Sandvik, http://www.mrlind.com</td>
			<td style="width:215px;"></td>
			<td>For oxidising silicon wafer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Small spot spectroscopic reflectometer</td>
			<td>e.g. Nanometrics NanoSpec 3000 reflectometer, www.nanometrics.com/</td>
			<td style="width:215px;"></td>
			<td>To measure depth of silicon dioxide layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silane adhesion promoter</td>
			<td>e.g. Merck Silane A174 adhesion promoter. Merck Chemicals, www.merck-chemicals.de/</td>
			<td style="width:215px;">1076730050</td>
			<td>Pre-applied to wafer to encourage parylene deposition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parylene-C&#160;</td>
			<td style="width:572px;">e.g. Ultra Electronics, www.ultra-cems.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCS Labcoter 2 deposition Unit, Model PDS2010</td>
			<td style="width:572px;">SCS equipment, Surrye, UK, www.scscoatings.com/</td>
			<td style="width:215px;">Model PDS2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexamethyldisilazane (HMDS) adhesion promoter</td>
			<td style="width:572px;">e.g. SpiChem, www.2spi.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Automated track system for dispensing photoresist on wafers.</td>
			<td>e.g SVG (silicon Valley Group) 3 inch photo-resist track,&#160;</td>
			<td style="width:215px;"></td>
			<td>Automated track system for dispensing photoresist on wafers. A prime oven bakes the wafer and dispenses the adhesion promoter, HMDS. A combination spinner dispenses photoresist.&#160; Pre-bake oven cures the resist.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Photo-resist: Rohm &#38; Haas</td>
			<td style="width:572px;">Rohm &#38; Haas, www.rohmhaas.com/</td>
			<td style="width:215px;">&#160;SPR350-1.2 positive photo-resist</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Phot-mask aligner</td>
			<td>e.g. Suss Microtech MA/BA8 mask aligner, www.suss.com</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microchem MF-26A developer</td>
			<td>Microchem MF-26A developer, www.microchem.com</td>
			<td style="width:215px;"></td>
			<td>Removes exposed reogions of photoresist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Plasma etch system</td>
			<td>e.g. JLS RIE80 etch system, JLS Designs, www.jlsdesigns.co.uk</td>
			<td style="width:215px;"></td>
			<td>Removes exposed regions of parylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wafer dicing saw</td>
			<td>e.g. DISCO DAD 680 Dicing Saw, DISCO Corporation, Japan, www.disco.co.jp</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone&#160;</td>
			<td style="width:572px;">e.g. Fisher Scientific, www.fishersci.com/</td>
			<td style="width:215px;">A929-4&#160;</td>
			<td>To wash off residual photoresist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen Peroxide&#160;</td>
			<td style="width:572px;">e.g. Sigma-Aldrich, www.sigmaaldrich.com</td>
			<td style="width:215px;">H1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">98% Sulphuric Acid</td>
			<td style="width:572px;">e.g. Sigma-Aldrich, www.sigmaaldrich.com</td>
			<td style="width:215px;">435589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum&#160;</td>
			<td style="width:572px;">Gibco-Invitrogen, www.invitrogen.com</td>
			<td style="width:215px;">10437</td>
			<td>Standard chip activation.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:572px;">Gibco-Invitrogen, www.invitrogen.com</td>
			<td style="width:215px;">14170</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell patterning on photolithographically defined 
parylene-c: sio2 substrates

细胞图案平台支持广泛的研究目标，如建设预定义的体外神经网络和细胞生理学的某些主要方面的探索。要轻松地结合细胞图案用多电极阵列（MEA）和技术的硅基&#39;在一个芯片上的实验室“，进行精微兼容协议是必需的。我们描述了利用了在SiO 2晶片的聚合物聚对二甲苯-C沉积的方法。光刻使的聚对二甲苯-C精确和可靠的图案在微米级的分辨率。通过浸渍在胎牛血清（或另一特定活化溶液）中的衬底的结果，其中培养的细胞粘附，或由聚对二甲苯或SiO 2区分别被击退后续激活。这种技术允许范围广泛的细胞类型（包括主要鼠海马细胞，HEK 293细胞系，人类神经元样畸胎的图案细胞系，原代鼠小脑颗粒细胞和​​原代人脑胶质瘤衍生的干细胞样细胞）。然而有趣的是，该平台是不通用的，反映了细胞特异性粘附分子的重要性。此细胞图案化工艺是成本有效的，可靠的，并且重要的是可结合到标准的微细加工（芯片制造）协议，集成微电子技术铺平了道路。

图案化的SiO 2用聚对二甲苯-C的光刻工艺示于图1。一旦准备，活化的胎牛血清的芯片能够广泛的文化图案化的细胞类型。我们小组已经成功地图案化的原代鼠海马细胞14-16的HEK 293细胞系17，人的神经元样畸胎癌（HNT）细胞系18，原代鼠小脑颗粒细胞和原代人脑胶质瘤来源的干细胞样细胞。 图3显示的HEK 293细胞的组成上以“十字型”圆形扩展节点的聚对二甲苯模式强大的图形。芯片激活在这个例子中是胎牛血清。与此相反， 图4示出了通过使用替代的活化溶液，以增加图案形成平台的潜力。使用牛血清白蛋白（3毫克/毫升）的溶液和<html>纤连蛋白（1微克/毫升）在HBSS中，前面的图案形成信条已经反转。 图5示出了不同的细胞类型（从高级别胶质瘤衍生的主要来自人的干细胞样细胞系）。这里，底层的图案影响细胞行为的几何形状，与图4A中所示的模式促进细胞生长过程中沿薄的聚对二甲苯-C跟踪， 如图4B和4C。 某些细胞类型使用已建立的胎牛血清激活协议时不要格局。 图6说明3T3 L1细胞成长至汇合与聚对二甲苯-C和SiO 2区之间没有明显细胞质，排斥或细胞质粘性差。 pload/50929/50929fig1.jpg“/&gt; 图1。流示出的过程中在SiO 2上的聚对二甲苯-C的模式制造图。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig2highres.jpg" src="/files/ftp_upload/50929/50929fig2.jpg" /> 图2示出了用于在芯片激活步骤图案化聚对二甲苯-C和SiO 2结构域的变化的接触角。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig3highres.jpg" src="/files/ftp_upload/50929/50929fig3.jpg" /> 图3。 HEK 293细胞后在体外三天parylene-C/SiO 2培养的活细胞成像。薯条温育3小时的胎牛血清后的细胞以5×10 4个细胞/ ml的浓度接种在悬浮液中。聚对二甲苯-C促进细胞黏附，而裸露的SiO 2排斥细胞。 <a href="https://www.jove.com/files/ftp_upload/50929/50929fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig4highres.jpg" src="/files/ftp_upload/50929/50929fig4.jpg" /> 图4。 HEK的活细胞成像293细胞培养于parylene-C/SiO 2后在体外 3天芯片3小时在不同的理性化活化激活解决方案：A：胎牛血清，B：玻连蛋白（1微克/毫升）在HBSS， C：牛血清白蛋白（3毫克/毫升）+玻连蛋白（1微克/毫升）在HBSS中，D：牛血清白蛋白（3毫克/毫升）+纤连蛋白（156克/毫升）的HBSS中。细胞接种是在悬浮液以5×10 4个细胞/ ml的密度。请注意如何处理不同的芯片，导致逆转先前的图案教条，与SiO 2的现在粘合剂和聚对二甲苯-C排斥的。聚对二甲苯-C节点直径250微米，改编自Hughes 等 17 <a href="https://www.jove.com/files/ftp_upload/50929/50929fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig5highres.jpg" src="/files/ftp_upload/50929/50929fig5.jpg" /> 图5。原代人脑胶质瘤来源的干细胞样细胞的免疫荧光图像上生长的聚对二甲苯-C上的SiO 2甲各种图案。示意示出在B和C所示的网状的聚对二甲苯设计</strong&gt; B：固定细胞的荧光显微照片（ 体外后4天）染色胶质纤维酸性蛋白（GFAP）C：活细胞在同一芯片上的光显微镜D：荧光图像说明在不同的聚对二甲苯GFAP染色细胞。设计E：该节点的反射图像并讲话聚对二甲苯设计成像在D中<a href="https://www.jove.com/files/ftp_upload/50929/50929fig5highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/50929/50929fig6highres.jpg" src="/files/ftp_upload/50929/50929fig6.jpg" /> 图6。 3T3 L1细胞体外后四天parylene-C/SiO 2培养的活细胞成像。芯片后，瓦特在胎牛血清3小时激活HICH细胞接种在悬浮液（3×10 4个细胞/ ml）。在这种情况下，该平台不使图案，与细胞变得同样融合于聚对二甲苯-C和SiO 2区。 <a href="https://www.jove.com/files/ftp_upload/50929/50929fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates at JoVE.com

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

本协议描述了一个微加工兼容的方法在SiO 2细胞图案。预定义聚对二甲苯-C设计光刻印刷在SiO 2晶片。温育后用血清（或其它活化溶液）细胞粘附具体地（和根据的符合性生长）相关的聚对二甲苯-C，而被排斥由SiO 2区。

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 ...

cell-patterning-on-photolithographically-defined-parylene-c-sio2

Research

JoVE Journal

Bioengineering

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

13.2K 次观看.  The University of Edinburgh. 本协议描述了一个微加工兼容的方法在SiO 2细胞图案。预定义聚对二甲苯-C设计光刻印刷在SiO 2晶片。温育后用血清（或其它活化溶液）细胞粘附具体地（和根据的符合性生长）相关的聚对二甲苯-C，而被排斥由SiO 2区。 

视频: Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

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

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 ...

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. ...

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, ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

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 ...

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.

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 ...

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...