Name: rapid subtractive patterning of live cell layers with a microfluidic probe
Uploaded: 2016-09-15T12:30:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe at JoVE.com

This work was supported by the European Research Council (ERC) Starting Grant, under the 7th Framework Program (Project No. 311122, BioProbe). We thank Dr. Julien Autebert and Marcel Buerge for technical assistance and discussions during the development of the protocol and the platform. Prof. Petra Dittrich (ETH Zurich), Prof. Bradley Nelson (ETH Zurich), Dr. Bruno Michel and Dr. Walter Riess are acknowledged for their continuous support.

1. MFP头部和平台的清理和准备注:此协议使用垂直取向硅钢玻璃混合MFP头15,16。头部的硅成分包含微通道，这是蚀刻的100微米的深度。蚀刻的硅阳极使用粘接接合到玻璃上。信道设计包括由六个信道，每两个用于注射和抽吸，和两个用于注入浸泡液体（ 图1）的图案。用于注射浸液渠道补充媒体围绕生物样品，这样可避免因误吸和蒸发损失。的内部通道，并在当前工作中使用的外通道是100×100微米，分别为200×100微米。得到后加工和加工，具有明确的渠道和抛光顶点MFP头。 <ol><li> 泵站的制备和流体处理仪器<ol><li>使用具有1/16&#39;&#39;（1.59 mm）外直径和0.02&#39;&#39;（0.51 MM）内径为连接到注射器所有管路毛细管。有所不同基于应用的毛细管尺寸，并获得相应的连接器和接头与注射器接口MFP头。无论稀释是必要使用超纯水。 </li><li>清洁注射器（250 - 500微升体积）和注射器活塞用超声波在超纯水之前于─0.5％的漂白粉溶液和后活细胞实验。用清水彻底冲洗。通过完全浸入其提示在水浴中，并使用活塞吸移用水填充。同时用注射器轴密封在注射器的出口接触水浴内清除液体。重复抽吸和吹扫直至没有气泡可见于注射器列。 </li><li>充式注射器连接到使用鲁尔锁连接器的注射泵。使用安装在一个开关阀泵以从针筒液体直接两个毛细管导致要么到MFP头或向液槽（ 图6）中的一个。 （如果没有切换阀可参见1.4.4）。在约10的流速吹扫从注射器与水既毛细血管 - 50微升/分钟，这取决于注射器的大小，直到大约10微升的水在注射器留。 </li><li>前插入清除毛细血管进入一个合适的微流体连接器/适配器，它在头部的通道孔接口（ 例如，M1连接器-看材料）。 </li></ol></li><li> MFP头准备 <ol><li>清洁通过超声处理使用标准清洗或0.5％的漂白剂为严格清洗玻璃器皿洗涤剂MFP的头部，5分钟。浸渍在水中的顶点和施加真空到通路清除与水通道。 </li><li>检查是否有潜在障碍（堵塞），并重新立体显微镜下渠道泥炭前面的步骤，如果必要的。 </li><li>装入清洁头的头支架和螺钉与预插入并吹扫油管到头部的连接器。螺钉的头部保持器与Z阶段，这是用于头和细胞单层之间的间隙距离的控制。 </li></ol></li><li> 校准MFP平台的扫描阶段 <ol><li>前连接头部到平台执行X，Y和Z级的端点校准，根据制造商的与适当的软件接口协议。校准阶段，确保在定位MFP头精度。 </li><li>获得通过将MFP头部比没有细胞室幻灯片粗零间隙距离（零），并在5微米的步骤慢慢下降。在与基板的探针接触，牛顿环应观察。这是一个粗略估计。一个准确的位置是调整探针顶点共面的substrat后所得到即</li><li>为了确保探头顶点的共面性，调整用测角器（在头的接口和Z台）的头部的倾斜。当形成确保牛顿环是对称的（ 图1）。移动至的MFP头20微米远离基底以及使用该测角器调整倾斜。重复的深浅，调零和倾斜调整，直至牛顿环都在接触对称的。随着倾斜调整，设置产生对称的牛顿环为零的z位置。 注:Z-阶段控制所述头-基板间距离，而XY台控制所述衬底的扫描（ 图2）。详细解释可以在我们早期的工作14被发现。 </li></ol></li><li> 化学药品制剂为当地清除细胞层 注意:在适当情况下，使用安全设备（ 如丁腈手套，防护眼镜）正在编制的化学品。使用通风呼d。如果必要的制备溶液。准备好所有的化学品和缓冲区用超纯水作为稀释剂。 <ol><li>制备的50mM NaOH溶液作为用于内注射通道（i2的与图1流量Q I 2）的处理液。 </li><li>制备用于外注射（I1与图1流量Q I1）所需的提取缓冲液，1mM的乙二胺四乙酸（EDTA），0.5％吐温20组成的，和10μM若丹明B在50mM Tris（羟甲基）氨基甲烷在pH值8.对于清洗，在抽吸注射器用水。 </li><li>使用过滤0.2微米的注射器过滤所有的解决方案。德加用干燥器直至溶气站浮出水面所有过滤解决方案。 </li><li>使用连接到注射泵漏极线，清除残余的水和吸出脱气溶液加入在40微升/分钟的注射针筒，直到注射器满（250或500微升）。这保证了注射器，连接器和毛细血管无气泡填充。注射器开关阀的双毛细管系统允许注射器中期试验的灌装。在不存在这样一个开关阀，断开从头部毛细血管和由它们重新连接到头部前抽吸脱气溶液填充毛细管和注射器。 </li><li>抽吸处理，并通过毛细血管屏蔽液体可在运行过程中使用小卷。如果化学物质可以在大量的准备，填写直接与所需的解决方案注射器。例如，含有水抽吸注射器可以使用这种方法来填补。 </li><li>清除毛细血管的MFP头分配在1.4.1和1.4.2定义的每个通道的液体。 注意:提取缓冲溶液屏蔽的NaOH在内部约束和在缓冲器中的若丹明组件便于日的可视化操作过程中êhHFC。 </li><li>如果细胞过度汇合，与出现在培养的表面上细胞聚集体，补充有10％蛋白酶K.提取缓冲这种补充的NaOH对这些聚集体由作为裂解物进入抽吸通道溶解变性的蛋白质的操作。这可以防止操作过程中的通道堵塞。 </li></ol></li><li> 细胞单层的上腔室玻片制备 注意:使用细胞培养罩培养细胞和处理设备按照由实验室的生物安全官员设置的规定。注意特定细胞系的特殊要求，并相应地调整协议和设备。 <ol><li>用于培养和细胞的扩展用细胞培养孵化器（在5％的CO 2，并在37℃），以进行图案化。使用标准的细胞培养协议17,18 T中瓶中进行扩张。用培养介质根据具体CEL的要求L线（ 如血清和抗生素补充DMEM培养MCF7和MDAMB 231）。 </li><li>关于在培养瓶中达到细胞汇合，trypsinize并收集细胞和种子2×10 5个细胞/ cm 2的各2室载玻片构图和培养过48小时的。使用该单元中的腔室作为用于细胞生长和存活的控制中的一个。 </li><li>上的玻片到达细胞汇合时，孵育细胞45分钟，用500μL细胞追踪染料溶液（ 例如 ，绿色- CMFDA或橙色- CMRA）在无血清培养基中制备10μM的浓度。这是图案化过程中对细胞的可视化实现。通过使用移液管轻轻地冲洗每一个腔室，并在图案形成实验血清的补充培养基随后培养细胞洗涤标记的细胞用PBS。 </li><li>获得小区跟踪染细胞表面的参考图像用于细胞计数的目的。这样做是为了确保细胞层的汇合。此外，它可以作为如果样品回收率和DNA分析是目标量化实验的辅助。 注意:如果该细胞活力，以图案化期间进行评估，所述细胞可以用在根据制造商的指令的活/死细胞毒性试剂盒进行染色。 </li></ol></li></ol> 2.创建层次水力流体密封（hHFC） <ol><li>移到细胞单层在MFP从载玻片50μm的间隙距离。同时确保hHFC的接触这个间隙距离也占单层表面和厚度变化。 </li><li>注入的NaOH在6或8微升/ I2通过闵。评估其他的流速（ 即，Q I1，Q A1和Q A2），使用流规则在图3中。 </li><li>通过使用注射针筒改变Q I2 / Q I1的比率调节内的HFC的大小。例如，使用一个Q为输入I1 1.3微升/分钟和4微升/分钟，用Q I2固定为8微升/分钟，这导致150的氢氧化钠足迹之间- 300细胞（100 - 200微米2 /细胞）（ 图3D）。 </li><li>注入从MFP上头部最外侧的孔完全培养基，在20微升/分钟的流速在hHFC的操作期间以考虑媒体和抽吸的蒸发。 </li></ol> 3.图案细胞单层使用hHFC 注意:扫描图案确定在何处将细胞中提取的细胞单层（减色图案形成）的区域，留下剩余的细胞，以研究特定生物的问题。这个图案可以是直线或点的阵列，例如。复杂的图案需要一个合适的扫描轨迹设计。例如，一个方格扫描轨迹提供小区区域的网格（ 图4A中所示，例如</strong&gt;的），这将使研究不同刺激的作用，对细胞在不同的方格，而在近距离内。这些模式可以用在平台上，在控制软件允许为MFP头在细胞单层扫描轨迹的脚本的XY阶段控制来创建。 <ol><li>设置阶段软件扫描探针头过在用户定义图案细胞单层（通过设定X，Y，Z坐标）留在50μm的间隙距离为10μm/ s的扫描速度。 </li><li>与嵌套hHFC运转中且在与单层接触，扫描的MFP具有所需图案的轨迹来实现图案化细胞的去除。 </li><li>用于第一小区类型除去后的共培养，种子不同细胞系以填补空白，使用在节1.5中描述的方法。嵌套和捏模式（通过增加间隙距离，例如）来控制细胞去除之间移动。 注:扫描速度可能必须investi门控为其他细胞系。在这种示范效应完整的细胞去除过度的使用细胞系的扫描区域使用的扫描速度。 </li></ol> 4.下游加工的取样和DNA扩增<ol><li>准备采样站的溶胞产物的下游分析。对于取样站，使用3D打印的8条PCR管持有人。可替代地，选择一个适当的管保持器，作为该适配器，其能够被安装在基板以外的头的扫描范围内。擦拭用70％乙醇或根据所需的应用程序的严格性等表面净化剂的管子保持架。使用的管子保持架的磁夹将其附加到衬底保持器。 </li><li>制备取样站后，放置从单层MFP的头100微米。开始用50mM NaOH溶液作为处理液与1流速，6内注入通道操作hHFC，-7和-17.5微升/分钟分别为Q为输入I1，Q I2，Q A1和Q A2。 <ol><li>一旦流体密封稳定（在大约10秒），下降探头至50μm的间隙距离，以执行与hHFC细胞的选择亚群的基于氢氧化钠本地裂解。一旦亚群的溶解完成（约30％足迹秒），指示头朝向在采样站的管子。弹出收集裂解物到PCR管中直接（ 图6）。 </li></ol></li><li>对于溶胞产物的下游处理中，首先由裂解物用Tris缓冲液（1:1）混合中和该溶液的pH值。中和后，加热该溶胞产物，以95°C下30分钟。然后将裂解物直接加载到一个标准的qPCR工作流由仪器供应商设置的。 </li></ol>

<ol>
	<li>Th&#233;ry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+as+a+tool+to+decipher+cell+morphogenesis+and+functions.">Micropatterning as a tool to decipher cell morphogenesis and functions.</a> J. Cell Sci. 123 (Pt 24), 4201-4213 (2010).</li><li>Goubko, C. A., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+multiple+cell+types+in+co-cultures:+A+review.">Patterning multiple cell types in co-cultures: A review.</a> Mater. Sci. Eng. C. 29 (6), 1855-1868 (2009).</li><li>Kaji, H., Camci-Unal, G., Langer, R., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+systems+for+the+generation+of+patterned+co-cultures+for+controlling+cell-cell+interactions.">Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.</a> Biochim. Biophys. Acta. 1810 (3), 239-250 (2011).</li><li>Cho, C. H., Park, J., Tilles, A. W., Berthiaume, F., Toner, M., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layered+patterning+of+hepatocytes+in+co-culture+systems+using+microfabricated+stencils.">Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.</a> Biotechniques. 48 (1), 47-52 (2010).</li><li>Sch&#252;tze, K., Lahr, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+expressed+genes+by+laser-mediated+manipulation+of+single+cells.">Identification of expressed genes by laser-mediated manipulation of single cells.</a> Nat. Biotechnol. 16 (8), 737-742 (1998).</li><li>Salazar, G. T. A., Wang, Y., 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=Micropallet+arrays+for+the+separation+of+single,+adherent+cells.">Micropallet arrays for the separation of single, adherent cells.</a> Anal. Chem. 79 (2), 682-687 (2007).</li><li>Guillaume-Gentil, O., Zambelli, T., Vorholt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+single+mammalian+cells+from+adherent+cultures+by+fluidic+force+microscopy.">Isolation of single mammalian cells from adherent cultures by fluidic force microscopy.</a> Lab Chip. 14 (2), 402-414 (2014).</li><li>Iwata, F., Adachi, M., Hashimoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+scraper+based+on+an+atomic+force+microscope+for+detaching+a+living+cell+from+a+substrate.">A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate.</a> J. Appl. Phys. 118 (13), (2015).</li><li>Riahi, R., Yang, Y., Zhang, D. D., Wong, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> J. Lab. Autom. 17 (1), 59-65 (2012).</li><li>Juncker, D., Schmid, H., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipurpose+microfluidic+probe.">Multipurpose microfluidic probe.</a> Nat. Mater. 4 (8), 622-628 (2005).</li><li>Kaigala, G. V., Lovchik, R. D., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+the+&amp;#34;open+space&amp;#34;+for+performing+localized+chemistry+on+biological+interfaces.">Microfluidics in the &amp;#34;open space&amp;#34; for performing localized chemistry on biological interfaces.</a> Angew. Chem. Int. Ed. Engl. 51 (45), 11224-11240 (2012).</li><li>Qasaimeh, M. A., Gervais, T., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+quadrupole+and+floating+concentration+gradient.">Microfluidic quadrupole and floating concentration gradient.</a> Nat. Commun. 2 (May), 464 (2011).</li><li>Christ, K. V., Turner, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+hydrodynamically+confined+microfluidics:+controlling+flow+envelope+and+pressure.">Design of hydrodynamically confined microfluidics: controlling flow envelope and pressure.</a> Lab Chip. 11 (8), 1491-1501 (2011).</li><li>Autebert, J., Kashyap, A., Lovchik, R. D., Delamarche, E., Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+hydrodynamic+flow+confinement:+efficient+use+and+retrieval+of+chemicals+for+microscale+chemistry+on+surfaces.">Hierarchical hydrodynamic flow confinement: efficient use and retrieval of chemicals for microscale chemistry on surfaces.</a> Langmuir. 30 (12), 3640-3645 (2014).</li><li>Kaigala, G. V., Lovchik, R. D., Drechsler, U., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vertical+Microfluidic+Probe.">A Vertical Microfluidic Probe.</a> Langmuir. 27 (9), 5686-5693 (2011).</li><li>Cors, J. F., Lovchik, R. D., Delamarche, E., Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+compact+and+versatile+microfluidic+probe+for+local+processing+of+tissue+sections+and+biological+specimens.">A compact and versatile microfluidic probe for local processing of tissue sections and biological specimens.</a> Rev. Sci. Instrum. 85 (3), 034301 (2014).</li><li>Pollard, J. W., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Cell Culture Protocols. , (1997).</li><li>Mitry, R. R., Hughes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cell+culture+protocols.">Human cell culture protocols.</a> Methods Mol. Biol. Springer. 531 (1), (2012).</li><li>Sakai, K., Ushida, T., Nagase, T., Nakamigawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cell+detachment+by+shear+stress.">Quantitative analysis of cell detachment by shear stress.</a> Mater. Sci. Eng. C. 17 (1-2), 55-58 (2001).</li><li>Rodriguez, N. M., Desai, R. A., Trappmann, B., Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+multicolor+dynamically+adhesive+substrates+to+control+cell+adhesion+and+multicellular+organization.">Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization.</a> Langmuir. 30 (5), 1327-1335 (2014).</li><li>Tasoglu, S., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+for+stem+cell+research.">Bioprinting for stem cell research.</a> Trends Biotechnol. 31 (1), 10-19 (2013).</li></ol>

The authors have nothing to disclose.

我们提出了一个通用的协议，用于细胞单层的减色图案化，使快速产生的空间限定单元图形。细胞单层的选择性裂解使用NaOH作为hHFC处理液进行的。该NaOH瞬时变性包含在细胞膜中的蛋白质。我们建议使用氢氧化钠50 mM的浓度，以保证裂解下游加工。该浓度可以为更快速细胞除去增加，提供裂解物下游加工是不是目的。在hHFC确保了细胞单层的未处理周围区域保持不受影响，并且可用于通过扩展图案或探测单元的其它性质。 细胞去除的速率是由流呈现的化学和剪切两者的函数。我们选择流率的操作范围有化学占主导地位的细胞去除。 20＆＃ - 10扫描速度181;米/使用的6氢氧化钠喷射流量秒 - 8微升/分钟用于促进&#39;快速&#39;减色图案化。细胞去除的速度迅速解决所面临的表面印刷了一些挑战基于图形化方法20,21。这些方法需要一个机制，以限制在空间上定义的区域的细胞的生长，例如，通过经由微接触印刷和细胞随后接种以获得图案细胞粘附蛋白的选择性沉积。它们是由低吞吐量的限制，并需要对图案生成几个连续步骤以及为每个图案的新模版/印章。所描述的方法并不需要上定义的区域的细胞的选择性生长，并通过执行相反印刷减色构图克服若干限制，同时不需要的细胞培养基材的任何额外的处理。 有了这个文件中提出的协议，吞吐量图案的细胞单层增加，同时获得所产生的图案的即时视觉反馈。这便于图案的实时修改。这样的控制可能在方案中的给定表面上的细胞活力是不均匀的关键。该方法的另一个优点是，相同的头部和化学可用于生成多个模式，从而减少了参与生成有图案的细胞单层的步数。 在头部的通道和头几何形状的尺寸可以根据应用的要求进行调整，从而使在图案形成的分辨率控制。使用的MFP头具有固定孔的尺寸与内孔在100×100微米的孔和侧孔，在200×100微米的进行在当前的工作所有实验中，具体。之所以选择这样的设计具有更大的外侧孔，以确保hHFC的连续操作而不被堵塞的孔的杂散细胞碎片。我们已经与内孔尺寸测试头50×50微米以及它们之间的50微米的间距，与细胞去除成功的结果。使用更小的孔，下降到10×10微米与10微米的间距，将允许缩放到更小的体积大小（〜30×30微米），从而使在图案形成更高的分辨率。我们观察到的业务问题与孔径尺寸小于10微米，由于从微粒堵塞。因为这样的操作上的困难，100微米是分辨率的电流限制，因此所描述的技术的限制。然而，我们也可以使用hHFC的液体成形能力解决这一问题。我们已经证明，细胞去除的分辨率可以通过控制Q I2 / Q I1被调谐为一组给定的光圈尺寸（ 图4b）和顶点到基板间隙10,14。在目前的工作中使用的阶段具有100nm的最小步长。这些控制参数的组合可以提高细胞去除在封装尺寸和扫描距离方面的空间分辨率。 如果图案化的共培养物所需的研究不同细胞类型之间的特异性细胞 - 细胞相互作用，可以使用所描述的协议来执行顺序的播种和图案化。最后，可从裂解物通过DNA中的单数峰获得的高质量扩增的DNA，如明显熔化曲线（ 见图7）。我们评估的周围1.6从单足迹（约300个细胞）使用qPCR，这是接近理论预期（6-8皮克/细胞）纳克的DNA量。这表示适于几个下游过程而避免使用任何DNA分离方法提取的DNA的量。这开辟了途径DNA的分析为基础的地方培养细胞的探测。 hHFC的塑造液体的能力也可用于沉积上交流活细胞和蛋白质tivated表面，其在与该协议提出的减色图案和顺序共培养组合能够对培养基材复杂的细胞和细胞 - 基质的图案的创建。多功能性和细胞单层的基于hHFC，消减图案和表演上提取的细胞DNA分析的可能性提供的控制，提供了一个强大的新工具设置为生物学家执行涉及细胞的相互作用空间分辨的研究。

在他们的本地环境中，细胞在生物组织中感知一系列生物化学和物理线索指导他们的成长，组织和发展。理解这些线索需要细胞 - 细胞和细胞 - 基质相互作用的选择性调查。这就需要对图案形成细胞单层方法的发展。方法培养（图案）几何独立不同的细胞类型的能在细胞生物学的物理和化学线索广泛的研究。最让图案细胞层当前的办法1 - 4取决于表面上沉积细胞粘附蛋白或使用微制造的模具在衬底上选择性生长。与此相反，在这里我们提出了一个方法来快速原位图案细胞单层， 即 ，将细胞在培养，通过在单层的选择区域中去除细胞。 9 usua -执行这样的减法图案5种方法LLY需要专门基板，表面处理，操作复杂，物理接触或消融使用激光，无意中影响活细胞。我们在这里使用的微流体探针（MFP）10,11，一个非接触式扫描微流体技术，流体动力地限制在衬底上的液体。在MFP中的一个重要组成部分是含微通道的微加工头（ 图1）。相关平台由注射泵为液体控制阶段的扫描控制和可视化和反馈（ 图2）的倒置显微镜。在其基本配置中，MFP头包括在顶点2微通道有孔，一个用于喷射处理液和其它用于与一些浸没液体（ 图3A）一起抽吸注入处理液。期间的MFP操作中，顶点是在从基板的固定距离。当吸流量（QA）是sufficiently比喷射流量（Q i）中 ， 也就是更高，Q 一个 : 数 Q i≥2.5，则处理液体被限制在衬底上。这导致了流体动力流动限制烃（HFC）。在其中处理液是在与基片直接接触的区域被称为足迹。对典型的操作条件时，处理液的HFC内的流的特征在于低的雷诺数（Re≈10 -2）和高的Peclet数（PE≈10 2）。这意味着流动液体在层流制度与对流是化学物种的质量传递的主要方式。流量限制数值和分析模型在别处描述12 - 14。 在本文中，我们使用的同时限制多个处理液体的方法，这就是所谓的分层HFC（hHFC）14。为了与MFP实现hHFC，二additiona需要升小孔提供注塑和愿望的次要来源。这使我们能够第二液体内限制一个液体。从内（处理）液体好处从杂散碎屑屏蔽由外（屏蔽）液体在衬底上。此外，hHFC允许以两种模式操作:（i）所述的嵌套模式，其中在所述内的HFC接触表面（ 图3B），和（ii）的捏模式，其中内液体失去处理液接触，并只有外液是在与基板（ 图3C）接触。两种模式之间的切换，用户可以影响或停止处理基板，并通过控制头-表面间隙或通过改变喷射流之间（Q i2的 / Q 的i1）的比率来实现的。对于给定的信道的几何形状，处理液的基板上的足迹可以通过调节流动条件（ 图3D）来控制。 HYDR钠氧化物氢氧化钠（NaOH）用作内处理液来裂解细胞，并且裂解物连续地从表面吸出。因为处理液的化学作用被定位于内的HFC足迹，相邻细胞保持未被干扰，这允许细胞 - 细胞相互作用的时空研究。 MFP的扫描功能允许（ 图4）的单元图形用户定义的几何形状的创建。此外，使用NaOH作为处理液的选择可容纳下游DNA分析（ 图7）。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Microfluidic Probe (MFP)</td><td></td><td></td><td>Silicon/glass hybrid probe head with channels patterned in silicon. Fabrication done in-house at IBM Research - Z&amp;uuml;rich</td></tr><tr><td>Lab Tek II Chamber slides with 2 chambers</td><td>ThermoFisher scientific, USA</td><td>154461</td><td>2 Chamber chamber slides for cell culture with each chamber providing an area of 4 cm&lt;sup&gt;2&lt;/sup&gt; and capable of holding up to 3 ml of media volume</td></tr><tr><td colspan="4">&lt;strong&gt;Chemicals and cell lines&lt;/strong&gt;</td></tr><tr><td>Sodium Hydroxide, Tris-Cl, Ethylenediaminetetraaceticacid, Rhodamine B</td><td>SigmaAldrich chemicals, USA</td><td>S5881, 252859,&amp;nbsp; E9884, R6626</td><td>Chemicals used for processing and shielding liquids</td></tr><tr><td>Proteinase K</td><td>Qiagen, Germany</td><td>19131</td><td>protease to digest denatuired proteins</td></tr><tr><td>Deconex 16 Plus</td><td>Bohrer Chemie, Switzerland</td><td></td><td>Universal cleaning agent for labaratory consumables. Used for non stringent cleaning.</td></tr><tr><td>DNA away&amp;nbsp;</td><td>ThermoFisher scientific, USA</td><td>7010</td><td>Surface decontaminant that denatures DNA and DNAases.</td></tr><tr><td>DMEM, high glucose, GLUTAMAX supplement</td><td>ThermoFisher scientific, USA</td><td>10566-016</td><td>Culturing medium for epithelial cells.</td></tr><tr><td>CellTracker Green CMFDA dye</td><td>ThermoFisher scientific, USA</td><td>C7025</td><td>Membrane permeant live cell labeling dye. Dye active for 72 hours.</td></tr><tr><td>CellTracker Orange CMRA dye</td><td>ThermoFisher scientific, USA</td><td>C34551</td><td></td></tr><tr><td>&amp;beta;-actin genomic primers for qPCR</td><td>Integrated DNA technologies, USA</td><td></td><td>Custom oligos used for DNA quality validation and qPCR.</td></tr><tr><td>MCF7 breast carcinoma cells</td><td>ATCC, USA</td><td>ATCC HTB-22</td><td>Cell lines used to produce co-cultures.</td></tr><tr><td>MDA-MB-231 breast carcinoma cells</td><td>ATCC, USA</td><td>ATCC HTB-26</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment and fluidic connections&lt;/strong&gt;</td></tr><tr><td>Motorized high precision stages</td><td>Custom machined components.&amp;nbsp; Linear axis motors from LANG GmBH, Germany</td><td>Customized linear axis stages from LT series</td><td>3 &amp;times; LT for 3 axes. LSTEP controller used for interfacing stages and PC through RS 232 port.</td></tr><tr><td>Syringes</td><td>Hamilton, Switzerland</td><td>1700 TLLX series</td><td>Interchangeable with syringes provided by other manufacturers with a 250-500 &amp;micro;l range.</td></tr><tr><td>Nemesys low pressure syringe pumps</td><td>cetoni GmbH, Germany</td><td></td><td>Component of pumping station.</td></tr><tr><td>Circular M1-connector</td><td>Dolomite microfluidics, United Kingdom&amp;nbsp;</td><td>3000051</td><td>Interface between vias in MFP head and tubing</td></tr><tr><td>Tilt/rotation stage Goniometer</td><td>OptoSigma, USA</td><td>KKD-25C</td><td>Goniometer to adjust coplanarity of MFP apex</td></tr><tr><td>DS Fi2 HD color camera (CCD)&amp;nbsp;</td><td>Nikon, Switzerland</td><td></td><td>Controlled using DS-U3 controller unit</td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>Win - Commander</td><td>LANG GmBH, Germany</td><td></td><td>Stage control software.</td></tr><tr><td>Qmix Elements</td><td>cetoni GmbH, Germany</td><td></td><td>Pump control software.</td></tr><tr><td>NIS Elements</td><td>Nikon, Switzerland</td><td></td><td>Basic research module for image acquisition and analysis.</td></tr></tbody></table>

rapid subtractive patterning of live cell layers with a microfluidic probe

微流体探头（MFP）有利于通过限制液体纳升体积进行生物基化学地方。利用一种特定实现MFP的，分层的流体动力流体密封（hHFC）的，多种液体在与基底接触的同时带来了。使用hHFC当地的化学作用和液体成型，被利用，被当地裂解和去除细胞创建小区模式。通过利用MFP的扫描功能，创建细胞单层的用户定义的模式。这个协议允许快速，实时和空间控制的小区图案形成，其可允许选择性细胞 - 细胞和细胞 - 基质相互作用的研究。

对细胞单层的快速减色图案形成所描述的协议是使用一个多组分的MFP平台（ 图1和2）证实。该协议采用分层流体动力流体密封（hHFC; 图3）局部治疗和用NaOH作为处理液体从单层细胞去除细胞。在hHFC构造包括内HFC和外HFC。在内部hHFC密闭的NaOH介绍了接触的细胞化学作用和剪切。在此足迹，将细胞均匀暴露于氢氧化钠的化学作用，由于内和具有可忽略的扩散的限制外的区域对流驱动的传质。关于另一方面，细胞的剪切，可以通过改变氢氧化钠的流速被改变。为了简化操作参数中，我们选择使化学作用细胞去除的主要机制相比于剪切。至估计的表面上由hHFC施加的剪切力，有限元模型是使用COMSOL多物理5.0构建的。模拟采用CFD模块的层流运行。被应用在当前示范使用流速和孔尺寸的范围内的两个入口边界条件，以几何的喷射孔和两个出口边界条件的抽吸孔（ 图5）。在所获得的模型时，流程规则所决定的剪切型面，而流率所限定的表面上的剪切的幅度。实际上，这两者的组合确定是否hHFC接触的表面上。牢记这些因素，我们开始寻找一个流量范围内进行操作，以获得化学占主导地位的细胞去除。要创建hHFC，我们总共使用愿望的注入3.5流速比的流规则。所使用的其他流规则被定义，以尽量减少抽吸通道内堵塞，这可以通过变性蛋白质附着在通道表面所引起的。使用开发的模型，我们发现流速为5〜10微升/分钟翻译到剪切应力1和3牛顿/米2之间。未经氢氧化钠的化学作用，例如在提取缓冲液的情况下，剪切应力不会高到足以除去细胞19。内观察到的范围内，我们注意到由于在低抽吸流速（ 即 ，Q I2 &lt;4微升/分钟），由于在通道中的细胞碎片的流动路径的扰动在较高流速在操作更加实用。 考虑所研究的剪切轮廓和实际的考虑，6和8微升/分钟的NaOH流速（Q I2）被用于图案形成实验和Q I1，Q A1和Q A2根据在图3所示的流规则。比注射流（Q I2 / Q I1）允许üs至进一步调节hHFC足迹（ 图3D和图4B）的大小，与由Autebert 等人 14阐述的基本原则。使用加上MFP平台的高分辨率扫描能力hHFC的液体整形的能力，我们展示了活细胞并网发电多尺度，进而表现出在发展联合培养（ 图4C）给定协议的应用程序。 该平台还使我们能够为下游分析样品进行裂解检索。来显示所获得的溶胞产物的质量，我们采样本地裂解的细胞从一到五的足印在两个独立的实验中，显示出在从裂解物（ 图7）得到的DNA的量的变化。在这里，我们使用扩增β-actin的引物（正向包含DNA的裂解液:GGATGCAGAAGGAGATCACT和反向:CGATCCACACGGAGTACTTG<html>使用4μl的在每个PCR反应的中和的裂解物）。 <img alt="图1" src="/files/ftp_upload/54447/54447fig1.jpg" /> 图1. 模块的MFP平台和头。（A）平台的业务模块包括电动注射器，电动平台和控制器。 MFP连接到机动化的Z阶段来控制头和衬底之间的间隙距离，和衬底保持器被连接到X和构成扫描系统的Y机动阶段。 （B）中的MFP的头具有流体通路连接泵站，安装孔安装头部上的Z阶段和渠道离开抛光顶点。顶点被设置共面的细胞培养基材。当顶点和基板是共面的，并在接触可以观察到的对称牛顿环。<html> p_upload / 54447 / 54447fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54447/54447fig2.jpg" /> 图2. MFP平台。高分辨率扫描平台配备有加工头保持器与高精度机动Z-工段接口。衬底支架被连接到XY台用于扫描目的。对于溶胞产物用于下游分析的恢复中，3D印刷采样站磁夹到衬底支架（在插图示出）的一侧。注射泵，倒置显微镜，控制器和显示器都位于平台周围。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3"SRC ="/文件/ ftp_upload / 54447 / 54447fig3.jpg"/&gt; 图3.分层流体动力流体密封（hHFC）的细胞去除的空间和时间的控制。（A）单HFC示意图。 （B）的嵌套和（C）hHFC操作捏模式。足迹两个不同的注射/抽吸流率（D）图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54447/54447fig4.jpg" /> 图4.使用MFP细胞单层模式。（A）单元网发电通过在MDA-MB-231细胞单层MFP的编程扫描。细胞用绿色细胞示踪染料染色。 （B）对于不同的喷射比的足迹（ 正）上的MCF7细胞单层。的示意图示出了与在正的变化内的HFC的形状的预期的变化。 （C）图案共培养紧跟着在减去地区播种MDA-MB-231细胞MCF7单层消减图案。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54447/54447fig5.jpg" /> 施加hHFC。上直线与内喷射流量的面增大的剪切应力时，表面上的图5的剪切应力 。最高剪切点被两个内孔（右下小图），其中，所述处理液体被限制（红色流线，左上小图）之间找到。在有限元模型中使用的孔径尺寸为200，100，100和200微米为I1，I2，A1和a2分别。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54447/54447fig6.jpg" /> 图6.在MFP平台的操作模式来执行细胞去除和图案化。用于通过细胞裂解减色图案形成的流路（A）的示意图。为简单起见，仅在每次注射和抽吸流路中的一个被示出。注射器中使用的是泵的排水阀填补。 i1和i2分别用于注射提取缓冲液和NaOH，。流路（B）的示意图用于裂解物回收。该流动路径被使用（A）中的流路的分析细胞裂解物的收集之后被激活。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig6large.jpg" target="_blank">请CLIC</a>ķ此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/54447/54447fig7.jpg" /> DNA的使用qPCR细胞裂解物图7.下游分析。在溶胞产物的DNA从5脚印（5 FP）和1足迹（1 FP）萃取的（A）的扩增曲线。对照裂解液收集这两种情况后提取。 （B）熔体从表示所提取的DNA的质量裂解物扩增的DNA的曲线。定量PCR进行（N = 2，N = 3）扩增β-actin的基因。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="人物" src="/files/ftp_upload/54447/54447figS.jpg" /> 图S1。通道设计的缩放图像的6声道MFP头执行hHFC用于形成图案的实验。频道是200，100，100，200微米宽和100微米深。在最外面的两个通道，及时补充浸液是500微米，宽100微米深。对于相同设计的一个GDS文件已被提供作为辅助这篇文章。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54447/54447figSlarge.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe at JoVE.com

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

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 ...

rapid-subtractive-patterning-live-cell-layers-with-microfluidic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.0K 次观看.  IBM Research - Zurich. 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.

视频: Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

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 ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

生物工程

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

神经科学

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

工程学

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

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 ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

活细胞层的快速消减带图案微流控探头

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微流体装置与Groove的模式，为研究细胞行为

图案的使用生物倒装芯片的胚胎干细胞

一种微流体装置研究多个不同的菌株

微流体技术，探讨细胞变形

微探针：操作和使用的局部表面处理

神经元共培养与单细胞精度的制备

通过顺序毛细管辅助组装对微生物和微粒进行图案化

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

图案蛋白质和细胞的通用的方法

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

活细胞层的快速消减带图案微流控探头

摘要

探索更多视频

此视频中的章节

微流体装置与Groove的模式，为研究细胞行为

图案的使用生物倒装芯片的胚胎干细胞

一种微流体装置研究多个不同的菌株

微流体技术，探讨细胞变形

微探针：操作和使用的局部表面处理

神经元共培养与单细胞精度的制备

通过顺序毛细管辅助组装对微生物和微粒进行图案化

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

图案蛋白质和细胞的通用的方法

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术