Name: fabricating complex culture substrates using robotic microcontact printing (r-µcp) and sequential nucleophilic substitution
Uploaded: 2014-10-31T15:00:00.000+00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ã‚ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

1.生成弹性邮票<ol style="margin-left: 40px;"><li>要生成PDMS印章的硅大师，采用计算机辅助设计软件设计的光罩的功能模式。 <ol><li>设计的第一图案为20×20阵列的环的具有300μm的内径（ID）和600微米的外径与1200微米的中心到中心的间距。 </li><li>设计第二图案作为20×20阵列的环带600微米的ID和900微米外径与1200微米的中心到中心的间距。 </li><li>此外，将1×1 平方毫米平方的参考标记在所有四个角的每个阵列的设计间隔1200微米的中心到中心的距离的角图案中的45°角。 </li><li>制造硅的主人用于本实验中，用1:1的长宽比，使用与微流体铸造别处15或协作详述标准光刻技术关联到一个300微米的特征深度。 注:功能深度小于100微米的可导致与基材表面接触之前，邮票异常变形。 注:此协议首先在已经得到的硅大师与光致抗蚀剂的叙述模式，这需要专门的设备和洁净室创建。这是最好的一个制作者或核心设备来创建这些图案高手请教。 </li></ol></li><li> Silanize硅大师O / N通过孵育（十三-1,1,2,2-tetrahydroocty）三氯硅烷的蒸气。 注:硅烷的蒸气有剧毒，只应在通风橱处理。 </li><li> 1比率的PDMS预聚物和PDMS在60℃下固化的硅烷化硅的主人的顶部剂在培养皿中O / N的:通过固化10创建的硅主人的逆复制品。 </li><li>从硅主人除去PDMS压模，并粘接邮票到丙烯腈 - 丁二烯 - 苯乙烯（ABS）的背衬或任何其他刚性材料S使用胶水。 注意:选择的材料不需要是透明的或生物相容的。但是，它必须与所述机器人的工具和更高的刚性比PDMS的接口提供一个平坦的表面。 </li></ol> 2.准备Coverslides <ol style="margin-left: 40px;"><li>漂洗显微镜coverslides在甲苯，甲醇和超声处理在丙酮中为下一个温和的氮气流中，用乙醇冲洗并干燥前1分钟（1 24毫米×50毫米＃）。 注:只有在通风橱处理甲苯和丙酮。 </li><li>外套coverslides以〜3.5 nm的钛（Ti），然后通过使用聚焦的电子束蒸发器18.0纳米的金（Au）。 注:此步骤是用各种金涂覆的基材包括硅和聚苯乙烯兼容。而有可能在现场产生这些，金涂覆的基材也可通过商业供应商。 注:钛层至少应为3纳米厚，而在衬底上的Au层应该是一个吨至少5纳米厚和范围可以全部在厚度为1mm取决于最终衬底16,17的理想的光学特性的方式。衬底不需要是用于制造光学透明的;然而，它便于制造基板的微观分析。 </li><li>在即将使用前冲洗镀金coverslides温和氮气下乙醇和干燥。 </li></ol> 3. R​​-μCP外环的<ol style="margin-left: 40px;"><li> R-μCP与选择标准的多关节机器人臂（SCARA）系统之前，校准使用系统软件的机器人工具效应和相应的双摄像头系统。 注意:这些工具被描绘在图1中。 </li></ol><img alt="图1" src="/files/ftp_upload/52186/52186fig1highres.jpg" /> 图1. R-μCP系统和机械臂工具。 </s仲&gt;（A）R-μCP系统的所有工装夹具大型看法，（一）真空吸盘，（二）试剂槽，（三）朝下的摄像头，（四）邮票嵌套夹具，（E）的机器人工具。 （B）中的机器人臂的工具描绘（六）金刚石尖蚀刻工具和（g）气动吸尘工具保持将a（h）的ABS支持的PDMS压模。 <ol start="2" style="margin-left: 40px;"><li>手动将600微米的ID和900微米OD突出的环在邮票嵌套夹具朝下一个PDMS印章。手动放置一个刚清洗干净的金涂覆coverslide上的真空吸盘，在图1A中显示的顶部，并使用该连接的实验室真空固定它。 </li><li>使用机械手的控制，对准朝下的相机在镀金coverslide的中心点，如在图2A中 。 注:此点最初可通过目视检查而定义的，并不需要很高的精度，由于PDMS压模得多LARG尔比的涂覆金的滑动。 </li><li>指示机器人臂以蚀刻四个参考的"X"标记，在一个正方形的尺寸为顶点3.8毫米由3.8毫米集中在使用连接到机器人臂的金刚石尖蚀刻工具的镀金coverslide。 （示于图2B;自动化的过程） 注:这将确保所有四个参考点都在朝下的摄像头之一的视觉框架。 </li><li>使用机器人气动吸工装，接机，并保持PDMS印章1毫米上面的邮票嵌套夹具， 如图2C。 （自动化过程） </li><li>利用朝上的摄像头和LED照明环描绘在图2D和E，可视化的印模的正方形的参考标记，并确定它们与照相机软件10倍，以确定所述时间戳的平均的X，Y，和从该中心的角度偏移机器人工装轴。 （自动化过程） </li><li>作为<strong&gt;图2A中，用面朝下的照相机来可视化和检测coverslide的蚀刻的基准标记的10倍，以确定coverslide的平均的X，Y中心位置和角度，从所述机器人的工具轴的中心偏移。 （自动化的过程）。 注意:一个内部计算由所述机器人系统的软件进行精确地对准印模的和盖玻片的X，Y中心位置和角度偏移量中的未来的R-微接触的步骤。 </li><li> 3.8）放置在链烷烃硫醇的ATRP引发剂，ω-mercaptoundecyl溴异丁酸乙酯（2毫摩尔的乙醇浴中的邮票），如在图2F中描绘。 （自动化过程） </li><li>从链烷醇溶液除去印模，并将其放置在加压到0.48巴（5 psi）的氮气流中蒸发乙醇， 如图2G。 1分钟后增加的氮气流的压力为1.03巴（15磅），以确保均匀的干燥。 （自动化流程） 注:不完全干燥会导致图案逼真的全部或部分损失。 </li><li>干燥后，将印模在镀金滑动的所计算出的中心位置，并降低其在100微米为增量，同时监测Z轴马达力， 如图2H所描绘。 注意:此连通的扭矩通过Z轴电机经历和在所述机器人指导软件显示。 （自动化过程） </li><li>停止降低一次的79.2千帕的预定压力已经达到印模，并保持与镀金的滑动，持续15秒的接触。 （自动化过程） 注:该压力值以下是目前邮票的设计和功能的高度优化。如果印模设计改变，特别为高纵横比的邮票，调整这些相应地通过尝试和错误的过程。 </li><li>慢慢地除去从镀金滑动印模和放置印章背面的印章嵌套夹具。 （自动化过程） </li></OL&gt; PEGMEMA对微模式Coverslides 4 SI-ATRP <ol style="margin-left: 40px;"><li>释放真空压力保持在微图案化coverslide，并将其转移到50毫升的Schlenk烧瓶中。用真空泵密封和脱气的Schlenk烧瓶中。 </li><li>添加5.5含大分子单体PEGMEMA（208.75毫摩尔），水（34.4毫升），甲醇（43.8毫升），铜（II）溴化物（1毫摩尔），和2&#39;，2-联吡啶（3毫摩尔）的原子转移自由基聚合反应混合物中的溶液在Schlenk烧瓶中。 </li><li>加入0.5毫升的L-抗坏血酸钠（454.3毫摩尔）的水，使反应开始，并允许它继续16小时，在室温下惰性气体。 注:在加入的L-抗坏血酸钠，反应颜色由浅绿色转变为暗棕色， 如图3A-B中。 注意:该反应可以继续超越16小时，但它不会显著增加的PEG刷子的长度。 </li></ol><imgALT ="图3"SRC ="/文件/ ftp_upload / 52186 / 52186fig3highres.jpg"/&gt; 图3.启动SI-ATRP的。（A）的反应和（B）以后的颜色变化如下添加L-抗坏血酸钠的启动。微图案化coverslide表面以下的SI-ATRP过程中（C）显微图像。比例尺1mm以下。 <ol start="4" style="margin-left: 40px;"><li>从申克烧瓶中取出微图案coverslide并用乙醇，水，以及乙醇和干燥下温和氮气流冲洗。 注:下面的SI-ATRP，表面修饰应肉眼可见的，并在显微镜下进行成像和分析， 如图3C所示 。 注:这是测试试验的精确度，因为它省却了免疫染色基板的最简单的方法。 </li></ol>的微模式PEGMEMA链5.叠氮化物官能<ol style="margin-left: 40px;"><li>放置在20毫升玻璃反应管形瓶中的微图案化coverslide。 </li><li>加入6毫升N，N-二甲基甲酰胺（DMF）中含有100mM氮化钠的反应小瓶。保持该反应在37℃下放置24小时。 注:只有在通风橱处理DMF。称出叠氮化钠时请务必小心。 </li><li>完成后，除去从反应小瓶的微图案化coverslide和冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 6.钝化溴官能PEGMEMA链<ol style="margin-left: 40px;"><li>放置在20毫升玻璃反应瓶微图案coverslide。 </li><li>加入6毫升含有100毫乙醇胺和300mM的三乙胺的反应小瓶二甲亚砜（DMSO）中的。保持该反应在40℃下放置24小时。 注意:只有在化学通风橱中处理的DMSO，乙醇胺，三乙胺等。 </li><li>完成后删除该微图案化coversl从反应小瓶的ide，冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 7. R-μCP内环和PEGMEMA的SI-ATRP <ol style="margin-left: 40px;"><li>开展R-μCP步骤如前面步骤3.2和3.6描述 - 3.12，与300微米的ID和600微米OD突出的环，使得具有较小的特点的环都放在前面微图案的环内代以PDMS印章。 注:此邮票的压力阈值是132.0千帕。如前面所提到的，这些压力设置为在该实验中所使用的印模特征优化。 </li><li>开展SI-ATRP步骤如前面的步骤描述4.1 - 4.4。 </li></ol>的微模式PEGMEMA链8.乙炔官能<ol style="margin-left: 40px;"><li>放置微图案coverslide在20毫升玻璃反应瓶中。 </li><li>加6毫升DMSO含100毫米炔丙基胺的反应小瓶。保持此反应混合物在室温放置24小时。 注:仅在通风橱处理DMSO和炔丙基胺。 </li><li>完成后除去从反应小瓶的微图案化coverslide和冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 9.铜催化的"点击"乙炔的生物素化终止PEGMEMA链<ol style="margin-left: 40px;"><li>放置微图案coverslide在20毫升玻璃反应瓶中。 </li><li>添加6毫升硫酸铜（15毫摩尔）/三[（1-苄基 -1H- 1,2,3-三唑-4-基）甲基]胺（TBTA，30毫摩尔）的（1:1体积/体积的水/ DMF）中含有562μM的叠氮基的PEG 4 -生物素偶联物的反应小瓶。在水中添加加入1.2ml L-抗坏血酸（0.15毫摩尔）至混合物中，以初始化该反应。 </li><li>通过10秒的反应液中的氮泡，用封口膜密封的小瓶，并24小时在室温下反应。 </li><li>当completion个反应瓶中取出微图案化coverslide，用清水冲洗干净，并放置在一个12孔聚苯乙烯菜。 </li></ol> 10.免疫荧光检测生物素化乙炔组<ol style="margin-left: 40px;"><li> 1小时在室温块DPBS（3％驴血清）微图案化coverslide。染色为生物素化的乙炔基团与链霉亲和546结合物（2微克/毫升）在DBPS（3％驴血清的PBS中）处理2小时，在室温。 </li><li>冲洗微图案coverslide 5次用DPBS 10分钟，温和搅拌。 注: 图4B中示出了滑动的图像后，此步骤就完成了。 </li></ol> 11.无铜"，点击"叠氮的生物素化终止PEGMEMA链注:如果需要的话，该底物修饰步骤可以在原位细胞培养过程中进行的。 <ol style="margin-left: 40px;"><li>离开微图案化coverslide在12孔Polystyrene盘以下DBPS​​冲洗。 </li><li>加2 ml DBPS（或细胞培养基）中含有20μM的DBCO-PEG 4 -生物素，并且允许该反应继续进行24小时，在室温（或37℃的培养箱）。 </li><li>完成后，冲洗微图案化coverslide 5次DPBS（或者干脆进行媒体的变化）。 </li></ol> 12.免疫荧光检测生物素标记的叠氮基团的<ol style="margin-left: 40px;"><li>染色为生物素化的叠氮基团与链霉亲和488结合物（2微克/毫升）在2毫升的DPBS（3％驴血清）培养2小时，在室温。 </li><li>冲洗微图案coverslide 5倍于DPBS中10分钟，温和搅拌。 </li><li>图象微图案coverslide使用共聚焦荧光显微镜。 图4A-C示出了该幻灯片的图片在此步骤之后完成。 注意:当进行组织培养试验用基板，跳过第10及该协议第12条。所需degre后ê官能化，通过在氮气流下漂洗两面用100％乙醇和烘干消毒工程coverslide。幻灯片转移到无菌的生物安全柜，并继续进行细胞接种和培养前冲洗幻灯片，用PBS五倍。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-precision+microcontact+printing+of+interchangeable+stamps+using+an+integrated+kinematic+coupling.">High-precision microcontact printing of interchangeable stamps using an integrated kinematic coupling.</a> Lab on a Chip. 11 (3), 455 (2011).</li><li>McNulty, J., 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-precision+robotic+microcontact+printing+(R-&#956;CP)+utilizing+a+vision+guided+selectively+compliant+articulated+robotic+arm.">High-precision robotic microcontact printing (R-&#956;CP) utilizing a vision guided selectively compliant articulated robotic arm.</a> Lab on a Chip. , (2014).</li><li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscalepatterning.">Soft lithography for micro- and nanoscalepatterning.</a> Nature Protocols. 5 (3), 491-502 (2010).</li><li>Nam, Y., Chang, J. C., Wheeler, B. C., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold-Coated+Microelectrode+Array+With+Thiol+Linked+Self-Assembled+Monolayers+for+Engineering+Neuronal+Cultures.">Gold-Coated Microelectrode Array With Thiol Linked Self-Assembled Monolayers for Engineering Neuronal Cultures.</a> IEEE Transactions on Biomedical Engineering. 51 (1), 158-165 (2004).</li><li>Ma, H., Wells, M., Beebe, T. P., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Initiated+Atom+Transfer+Radical+Polymerization+of+Oligo(ethylene+glycol)+Methyl+Methacrylate+from+a+Mixed+Self-Assembled+Monolayer+on+Gold.">Surface-Initiated Atom Transfer Radical Polymerization of Oligo(ethylene glycol) Methyl Methacrylate from a Mixed Self-Assembled Monolayer on Gold.</a> Advanced Functional Materials. 16 (5), 640-648 (2006).</li><li>Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem-cell+niche+as+an+entity+of+action.">The stem-cell niche as an entity of action.</a> Nature. 441 (7097), (2006).</li><li>Codelli, J. A., Baskin, J. M., Agard, N. J., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-Generation+Difluorinated+Cyclooctynes+for+Copper-Free+Click+Chemistry.">Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry.</a> Journal of the American Chemical Society. 130 (34), 11486-11493 (2008).</li><li>Debets, M. F., van Berkel, S. S., Schoffelen, S., Rutjes, F. P. J. T., van Hest, J. C. M., van Delft, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aza-dibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+2)+cycloaddition.">Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition.</a> Chemical Communications. 46 (1), 97 (2010).</li><li>DeForest, C. A., Polizzotti, B. D., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+click+reactions+for+synthesizing+and+patterning+three-dimensional+cell+microenvironments.">Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments.</a> Nature Materials. 8 (8), 659-664 (2009).</li><li>Roth, E. A., Xu, T., Das, M., Gregory, C., Hickman, J. J., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+printing+for+high-throughput+cell+patterning.">Inkjet printing for high-throughput cell patterning.</a> Biomaterials. 25 (17), 3707-3715 (2004).</li><li>Xu, T., Zhao, W., Zhu, J. M., Albanna, M. Z., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials.">Biomaterials.</a> Biomaterials. 34 (1), 130-139 (2013).</li><li>Brouzes, E., 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=Droplet+microfluidic+technology+for+single-cell+high-throughput+screening.">Droplet microfluidic technology for single-cell high-throughput screening.</a> Proceedings of the National Academy of Sciences. 106 (34), 14195-14200 (2009).</li><li>Meitl, M. 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=Transfer+printing+by+kinetic+control+of+adhesion+to+an+elastomeric+stamp.">Transfer printing by kinetic control of adhesion to an elastomeric stamp.</a> Nature Materials. 5 (1), 33-38 (2005).</li></ol>

The authors declare that they have no competing financial interest.

理想的基质用于组织工程将仿生并由此概括的天然组织中发现的关键的生物活性配体的空间分布。他们也将具有动态属性，使配体的时间调整，并且在它们出现，以允许定向组织形态发生和空间上的限制诱导细胞命运的分布模式。这种基材的制造需要在衬底上复杂和高度有序取向多种生化线索的固定。虽然模拟细胞的小生境所有的内源性因素在体外是不合理的，这里所描述的R-μCP系统允许?...

的PEG接枝表面显示共价结合的配体的生化，同时保持固有的不污性能的能力，使他们为工程定制微观环境对培养基质1,2,3的理想选择。通过配体结合的PEG介导刷的生物特异性相互作用使得在单个细胞的表型体内组织的微环境中的复杂的生物化学发现线索的作用还原论分析。此外，生物正交的"点击"化学方法可用来促进配体的定向固定化，使得它们在天然构象4-6给出。因此，PEG的微观空间的图案笔刷是一个多功能的工具来创建设计师在体外龛调查引起的固定生化线索6,7细胞信号转导。 产生生化立方米的空间格局的常用方法ES限嗣继承微接触印刷（μCP）镀金基板与PEG结合的烷基硫醇模式。然后，PEG-聚乙二醇化复合链烷硫醇的微图案化的自组装单分子层限制了生化分子， 例如，蛋白质的物理吸附，只在基板8,9的非图案区域。然而，通过这种技术产生的自组装是在长期的细胞培养介质中对氧化敏感。因此，μCP&#39;d硫醇自组装膜通常还采用表面引发的原子转移自由基聚合（SI-ATRP），以增加该地区的不污稳定性10接枝的PEG聚合物刷。具体地，微接触的烷基硫醇聚合引发剂，ω-meraptoundecyl溴异丁酸乙酯，对金涂覆的表面，随后由SI-ATRP的聚（乙二醇）甲基醚甲基丙烯酸酯（PEGMEMA）单体生成具有微图案化的长期，稳定，非表面结垢PEG刷。此外，这些<html>能够被进一步修改，以呈现不同的化学部分11。 以这个属性，沙等优势。人 。开发出一种方法来设计培养基质多组分PEGMEMA画笔呈现直角"点击"化学物质。在该方法中，他们使用了一系列的穿插顺序叠氮化钠，乙醇胺微接触/ SI-ATRP的步骤，和炔丙基胺的亲核取代来创建培养基材呈现多种固定化配体6的微型图案。而使用这样的化学品在与人工微接触结合来设计新的培养基材的电位是巨大的，它是由精度和准确度与多个微接触的步骤可以在一个单一的基片对齐的限制。将需要的精度和准确度水平高可再现地制造复杂中使用这些通用的技术体外龛。 e_content"&gt;为了解决此限制，几个自动化和半自动化的微接触系统已经生成。轮穴等人开发出了微接触系统，其中定制邮票被放置在轨道系统和进入共形接触以使用金包被的载玻片一个计算机控制的气动致动器，然而，这种方法需要的定制邮票设计的精确制造，并报告有10微米的精度与执行多个微接触步骤12，当所取得的精确度的报告。最近，利用集成的运动耦合系统的方法小于1微米的精度报告使用单个图案，但是无法准确地对准多个图案由于缺少精确控制从模具印模特征到模具13上 。另外，两者的以前的方法所需要的基底图案形成步骤之间保持固定从而显著限制性的表面改性化学，可以是分集利用。在这里，我们描述了一种自动化的R-微接触系统能够对多个微接触步骤，准确和精确的对准，同时允许在邮票的设计和制造的灵活性最大。此外，图案化衬底可以重复从冲压件之间的体系中除去，从而允许使用各种不同的底物改性化学品，包括顺序的亲核取代。衬底使用这样的化学工程已被用于预先细胞培养由我们两人6,14和其他7。因此，我们已经合并的R-微接触和连续的亲核取代反应来开发用于培养基材的复杂和微图案化的生化线索可伸缩的制造方法。

<table><tbody><tr><td>SCARA&amp;nbsp;</td><td>Epson</td><td>LS3-401ST</td><td>Higher end models with increased precision are available if desired.&amp;nbsp;</td></tr><tr><td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane</td><td>Gelest</td><td>SIT8174.0</td><td>CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesive Co</td><td>NC9020938</td><td>Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td></tr><tr><td>24 mm x 50 mm #1 Cover Glass Slides</td><td>Fisher Scientific</td><td>48393106</td><td>These can be purchased from a number of suppliers with varying dimensions to suit need.</td></tr><tr><td>CHA-600 Telemark Electron Beam Evaporator</td><td>Telemark</td><td>SEC-600-RAP</td><td>Requries specialized training.</td></tr><tr><td>EPSON LS3 SCARA</td><td>EPSON</td><td>LS3-401ST</td><td></td></tr><tr><td>&amp;omega;-mertcaptoundecyl bromoisobutyrate</td><td>Prochimia</td><td>FT 015-m11-0.2</td><td>Store at -20 &amp;deg;C. Other ATRP initiators may be used as this R-&amp;mu;CP platform is applicable to all micropatterning modalities.&amp;nbsp;</td></tr><tr><td>Schlenk Tube Flask 50 ml</td><td>Synthware</td><td>60003-078</td><td>Requires rubber stoppers with diaphram.</td></tr><tr><td>Poly(ethylene glycol) methyl ether methacrylate</td><td>Sigma Aldrich</td><td>447943</td><td>Shipped containing MEHQ and BHT free readical inhibitors.</td></tr><tr><td>Methanol (Certified ACS)</td><td>Fisher Scientific</td><td>A412-4</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Copper(II) Bromide</td><td>Sigma Aldrich</td><td>437867</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>2,2&#039;-Bipyridine</td><td>Sigma Aldrich</td><td>D216305</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>Sodium L-Ascorbate</td><td>Sigma Aldrich</td><td>A4034</td><td></td></tr><tr><td>20 ml Borosilicate Glass Scintillation Vials</td><td>Fisher Scientific</td><td>03-340-4E</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>S2002</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethyformamide</td><td>Sigma Aldrich</td><td>227056</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Ethanolamine</td><td>Sigma Aldrich</td><td>398136</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>T0886</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma Aldrich</td><td>276855</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Propargylamine</td><td>Sigma Aldrich</td><td>P50900</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>200 Proof Ethanol</td><td>University of Wisconsin Material Distribution Services</td><td>2292</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Azide-PEG3-Biotin</td><td>ClickChemistryTools</td><td>AZ104-100</td><td>Solubilized in DMF</td></tr><tr><td>Copper(II) Sulfate</td><td>Sigma Aldrich</td><td>C1297</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td><td>Sigma Aldrich</td><td>678937</td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma Aldrich</td><td>A7506</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Invitrogen</td><td>14190144</td><td></td></tr><tr><td>Donkey Serum</td><td>Sigma Aldrich</td><td>D9663</td><td>Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (&lt;em&gt;e.g.&lt;/em&gt; BSA) are available and serve this purpose.</td></tr><tr><td>12-Well Polystyrene Plate</td><td>Thermo Scientifit - NUNC</td><td>07-200-81</td><td>Plates can be purchased form a number of suppliers with varying dimensions.</td></tr><tr><td>DBCO-PEG4-Biotin</td><td>Clickchemistytools</td><td>A105P4-10</td><td>Solubilized in DMF</td></tr><tr><td>Streptavidin, Alexa Fluor 488 Conjugate</td><td>Life Technologies</td><td>S-11223</td><td>Solubilized in PBS</td></tr><tr><td>Streptavidin, Alexa Fluor 546 conjugate</td><td>Life Technologies</td><td>S-11225</td><td>Solubilized in PBS</td></tr><tr><td>Nikon A1-R Confocal Microscope</td><td>Nikon</td><td>Nikon Eclipse Ti, A1R</td><td>An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td></tr></tbody></table>

fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

使用手动调整μCP技术工程师培养基质与功能化正交PEG接枝画笔数组"点击"化学品已被证明在以往的工作6。然而，这提供的图案取向最小控制和通常导致功能化区域的重叠。这里，一个新颖的R-微接触系统被用于克服此限制，并且它能够准确图案的聚乙二醇刷的环用300微米的ID和600μm的外径呈现末端炔基的聚乙二醇刷的环单独的阵列中与600微米的阵列ID和900微米OD呈现终端叠氮基团被证实...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ã‚ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

10.4K 次观看.  University of Wisconsin, Madison. Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. 

视频: Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- µCP and Sequential Nucleophilic Substitution

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

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 ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

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. ...

生物学

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

3D 打印在微型神经元细胞培养设备中的应用

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

作者聚焦:用于分析大脑功能的模块化神经元网络

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

微尺度神经元细胞培养装置的双光子聚合3D打印

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...