作者承认包括英国工程和自然科学研究理事会在内的各种来源提供的财政支持 (赠款裁判。EP/K011685/1, EP/K024485/1) 和奖学金毕业生;Leverhulme 信托基金 (RPG-2014-292);威康信托机构战略支助基金 (105610/z/14/z);英国的理事会 (216196834);和曼彻斯特大学曼彻斯特大学研究所 (UMRI 泵启动基金) 和总统博士奖学金的 SW. 李博士 (Nanosurf AG) 的技术援助也得到了赞赏。

<ol>
	<li>Saavedra, H. M., 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=Hybrid+Strategies+in+Nanolithography.">Hybrid Strategies in Nanolithography.</a> Rep. Prog. Phys. 73, 036501 (2010).</li><li>Gates, B. D., 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=New+Approaches+to+Nanofabrication:+Molding,+Printing,+and+Other+Techniques.">New Approaches to Nanofabrication: Molding, Printing, and Other Techniques.</a> Chem. Rev. 105, 1171-1196 (2005).</li><li>Garcia, R., Knoll, A. W., Riedo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Scanning+Probe+Lithography.">Advanced Scanning Probe Lithography.</a> Nat. Nanotechnol. 9, 577-587 (2014).</li><li>Piner, R. D., Zhu, J., Xu, F., Hong, S., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Dip-Pen"+Nanolithography.">"Dip-Pen" Nanolithography.</a> Science. 283, 661-663 (1999).</li><li>Salaita, K., Wang, Y., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Dip-Pen+Nanolithography.">Applications of Dip-Pen Nanolithography.</a> Nat. Nanotechnol. 2, 145-155 (2007).</li><li>Lewis, 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=Fountain+Pen+Nanochemistry:+Atomic+Force+Control+of+Chrome+Etching.">Fountain Pen Nanochemistry: Atomic Force Control of Chrome Etching.</a> Appl. Phys. Lett. 75, 2689-2691 (1999).</li><li>Kim, K. -. H., Moldovan, N., Espinosa, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nanofountain+Probe+with+Sub-100+Molecular+Writing+Resolution.">A Nanofountain Probe with Sub-100 Molecular Writing Resolution.</a> Small. 1, 632-635 (2005).</li><li>Gruter, R. R., Voros, J., Zambelli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FluidFM+as+a+Lithography+Tool+in+Liquid:+Spatially+Controlled+Deposition+of+Fluorescent+Nanoparticles.">FluidFM as a Lithography Tool in Liquid: Spatially Controlled Deposition of Fluorescent Nanoparticles.</a> Nanoscale. 5, 1097-1104 (2013).</li><li>Salaita, K., 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=Massively+Parallel+Dip-Pen+Nanolithography+with+55+000-Pen+Two-Dimensional+Arrays.">Massively Parallel Dip-Pen Nanolithography with 55 000-Pen Two-Dimensional Arrays.</a> Angew. Chem. Int. Ed. 45, 7220-7223 (2006).</li><li>Huo, F., 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=Polymer+Pen+Lithography.">Polymer Pen Lithography.</a> Science. 321, 1658-1660 (2008).</li><li>Shim, W., 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=Hard-Tip,+Soft-Spring+Lithography.">Hard-Tip, Soft-Spring Lithography.</a> Nature. 469, 516-520 (2011).</li><li>Liao, X., Braunschweig, A. B., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Force-Feedback"+Leveling+of+Massively+Parallel+Arrays+in+Polymer+Pen+Lithography.">"Force-Feedback" Leveling of Massively Parallel Arrays in Polymer Pen Lithography.</a> Nano Lett. 10, 1335-1340 (2010).</li><li>Wang, S., Hosford, J., Heath, W. P., Wong, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Area+Scanning+Probe+Nanolithography+Facilitated+by+Automated+Alignment+of+Probe+Arrays.">Large-Area Scanning Probe Nanolithography Facilitated by Automated Alignment of Probe Arrays.</a> RSC Adv. 5, 61402-61409 (2015).</li><li>Noh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Multipoint+Force+Feedback"+Leveling+of+Massively+Parallel+Tip+Arrays+in+Scanning+Probe+Lithography.">"Multipoint Force Feedback" Leveling of Massively Parallel Tip Arrays in Scanning Probe Lithography.</a> Small. 11, 4526-4531 (2015).</li><li>Dalby, M. 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=The+Control+of+Human+Mesenchymal+Cell+Differentiation+Using+Nanoscale+Symmetry+and+Disorder.">The Control of Human Mesenchymal Cell Differentiation Using Nanoscale Symmetry and Disorder.</a> Nat. Mater. 6, 997-1003 (2007).</li><li>Connell, C. D., Higgins, M. J., Moulton, S. E., Wallace, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-Bioelectronics+via+Dip-Pen+Nanolithography.">Nano-Bioelectronics via Dip-Pen Nanolithography.</a> J. Mater. Chem. C. 3, 6431-6444 (2015).</li><li>Giam, L. R., 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=Scanning+Probe-Enabled+Nanocombinatorics+Define+the+Relationship+Between+Fibronectin+Feature+Size+and+Stem+Cell+Fate.">Scanning Probe-Enabled Nanocombinatorics Define the Relationship Between Fibronectin Feature Size and Stem Cell Fate.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 4377-4382 (2012).</li><li>Eichelsdoerfer, D. 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=Large-Area+Molecular+Patterning+with+Polymer+Pen+Lithography.">Large-Area Molecular Patterning with Polymer Pen Lithography.</a> Nat. Protoc. 8, 2548-2560 (2013).</li><li>Aran, K., Sasso, L. A., Kamdar, N., Zahn, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible,+Direct+Bonding+of+Nanoporous+Polymer+Membranes+to+PDMS+or+Glass+Microdevices.">Irreversible, Direct Bonding of Nanoporous Polymer Membranes to PDMS or Glass Microdevices.</a> Lab Chip. 10, 548-552 (2010).</li><li>Liao, X., Braunschweig, A. B., Zheng, Z., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-+and+Time-Dependent+Feature+Size+and+Shape+Control+in+Molecular+Printing+via+Polymer-Pen+Lithography.">Force- and Time-Dependent Feature Size and Shape Control in Molecular Printing via Polymer-Pen Lithography.</a> Small. 6, 1082-1086 (2010).</li><li>Sheehan, P. E., Whitman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol+Diffusion+and+The+Role+of+Humidity+in+"Dip+Pen+Nanolithography".">Thiol Diffusion and The Role of Humidity in "Dip Pen Nanolithography".</a> Phys. Rev. Lett. 88, 156104 (2002).</li><li>Geissler, M., 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=Fabrication+of+Metal+Nanowires+Using+Microcontact+Printing.">Fabrication of Metal Nanowires Using Microcontact Printing.</a> Langmuir. 19, 6301-6311 (2003).</li><li>Gmeiner, B., Leibl, H., Zerlauth, G., Seelos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Binding+of+Distinct+Functional+Fibronectin+Domains+to+Immobilized+Metal+Chelates.">Affinity Binding of Distinct Functional Fibronectin Domains to Immobilized Metal Chelates.</a> Arch. Biochem. Biophys. 321, 40-42 (1995).</li><li>Curran, J. M., 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=Introducing+Dip+Pen+Nanolithography+as+a+Tool+for+Controlling+Stem+Cell+Behaviour:+Unlocking+the+Potential+of+the+Next+Generation+of+Smart+Materials+in+Regenerative+Medicine.">Introducing Dip Pen Nanolithography as a Tool for Controlling Stem Cell Behaviour: Unlocking the Potential of the Next Generation of Smart Materials in Regenerative Medicine.</a> Lab Chip. 10, 1662-1670 (2010).</li><li>Davey, M. R., Anthony, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Cell Culture: Essential Methods. , 153-172 (2010).</li><li>Brinkmann, F., 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=Interdigitated+Multicolored+Bioink+Micropatterns+by+Multiplexed+Polymer+Pen+Lithography.">Interdigitated Multicolored Bioink Micropatterns by Multiplexed Polymer Pen Lithography.</a> Small. 9, 3266-3275 (2013).</li><li>Senesi, A. J., Rozkiewicz, D. I., Reinhoudt, D. N., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose-Assisted+Dip-Pen+Nanolithography+of+Oligonucleotides+and+Proteins.">Agarose-Assisted Dip-Pen Nanolithography of Oligonucleotides and Proteins.</a> ACS Nano. 3, 2394-2402 (2009).</li><li>Carnally, S. A. M., Wong, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+Catalysis+to+Enhance+Scanning+Probe+Nanolithography.">Harnessing Catalysis to Enhance Scanning Probe Nanolithography.</a> Nanoscale. 6, 4998-5007 (2014).</li></ol>

对齐算法和软件由曼彻斯特大学开发, 并且是专有的。可在 http://www.click2go.umip.com/下载。

该协议为用户提供了一种方便的方法, 可以快速地实现高均匀性和可控制的功能大小 (cm2) 区域的 nanolithographic 模式。这些大面积 nanopatterns 的衬底可以为各种应用进一步阐述。这项技术的一个主要应用是在 nanofabricated 表面进行细胞表面相互作用研究。本报告展示了这些材料的细胞培养的一些例子, 展示了 nanofabricated 基质对 hMSC 形态的控制。
此协议的关键启用程序是对齐过程 (步骤 4) 的自动化, 它允许在表面上高度均匀和高通量的功能生产, 降低到纳米尺度分辨率, 从而实现细胞培养实验的快速周转。采用这种对准算法进行的聚合物笔光刻能够在大约30分钟内产生纳米尺度特征。自动对齐的再现性和准确性, 从而使图案化特征的一致性, 但严重依赖于所产生的探针阵列的质量 (步骤1和 2)。在准备过程中产生钝、折断或缺失探针的任何缺陷;如陷气气泡 (步骤 1.5) 或不正确的分离探针从主 (步骤 1.8) 可能导致不准确的对准和质量差光刻。
此报告方法与依赖于强制反馈的其他对齐方法共有一个限制。准确地确定探针与表面接触时, 由于需要考虑环境环境和样品阶段的运动引起的背景振动而受到限制。一般而言, 传感器在µN (本例中为2µN) 中具有强制灵敏度, 但对齐算法的设计目的是只注册至少490µN 的力作为探头和表面之间的最终接触, 以避免任何 "误报" 结果从背景噪音。13因此, 这种方法倾向于产生大的特征 (1-2 µm), 因为探头必须在z轴上扩展一个很大的距离 (随之而来的是更高的力), 以便注册接触。为了补偿, 更小的功能可以通过减少光刻步骤中的z轴距离来生成 (例如, 在步骤5.2.3.2 中输入 "黑色" 设置, 而不是5µm)。
尽管如此, 即使有了这一限制, 自动化算法也能够解决并行扫描探针光刻方法的应用中的一个关键方面, 因为对齐以前是最需要时间和不精确的步骤。这些技术的实施。这种自动化现在将制造过程的速率限制步骤从对齐到平版书写本身。虽然本协议演示了这种对齐程序在 PPL 中的应用, 但该框架可以应用于一些 SPL 技术, 如脂质 DPN26和矩阵辅助光刻27以及未来潜在的催化探头系统。28

纳米技术的进展依赖于能够高效、可靠地制造表面纳米尺度特征的技术的发展。1,2然而, 在大面积 (多 cm2) 上可靠地产生这样的特性, 而且成本相对较低, 这是一个非平凡的努力。大多数现有的技术, 从半导体工业, 依靠烧蚀光刻制造 ' 硬 ' 材料。最近, 从扫描探针显微镜 (SPM) 中获得的光刻技术已经成为纳米设计快速成型的一种方便、通用的方法。3基于 SPM 的技术能够方便快捷地 "编写" 任何用户定义的模式。最知名的这些是蘸笔光刻 (DPN), 率先由 Mirkin等人,4在那里扫描探针被用作 ' 钢笔 ' 转移分子 ' 墨水 ' 的表面产生的特点, 类似于写作。在环境条件下, 当探针在表面扫描时, "墨水" 分子通过在探针和表面之间形成的水半月板转移到表面 (图 1)。DPN 从而允许 nanolithographic 沉积广泛的材料, 包括 ' 软 ' 材料, 如聚合物和生物分子。nanopipettes 还报道了5项相关技术, 这些方法使用了用管道进行流体输送的探头, 不同的称为 ' ' ' ' 和 ' 纳米喷泉笔 '。6,7,8
更广泛地应用 SPM 衍生光刻的主要障碍是吞吐量, 因为它需要过长的时间, 以一个单一的探针模式厘米尺度区域。早期致力于解决这个问题的重点是基于悬臂式 DPN 的并行化, 同时报告了 "一维" 和 "二维" (2D) 探针阵列, 用于对厘米大小的区域进行光刻。5,9然而, 这些悬臂阵列是通过相对复杂的多步骤制造方法产生的, 而且相对脆弱。聚合物笔光刻 (PPL) 的发明解决了这个问题, 通过更换标准的 SPM 悬臂与2D 阵列软硅氧烷弹性探头粘结到玻璃滑梯。10这一简单的探头设置大大降低了阵列大面积的成本和复杂性, 为更广泛的应用开辟了光刻。这种无悬臂结构也被扩展到硬尖端软弹簧光刻,11提供了一个混合软弹性支承与硬硅小贴士, 使改进的分辨率相比, 使用软弹性体提示。
执行这些2D 阵列技术的一个关键因素是探针阵列必须与表面基底完全平行, 以便在使用光刻时, 所有探头同时与表面接触。即使是一个小的错位也会导致从阵列的一侧到另一个的特征大小有很大的差别, 因为一些探针会在阵列下降的时候与表面接触, 而另一些探头会在以后接触到或根本不出现。12精确对准是特别重要的, 由于软弹性体探头的变形性, 在那里接触表面的探头将被压缩, 留下更大的足迹在表面上。
早期的研究人员使用纯粹的视觉检查来指导对准过程, 利用在阵列上方的摄像头观察金字塔探针的变形, 因为它们接触到了表面。10对齐方式是通过观察探针的哪一侧接触到表面首先, 然后调整角度和重复该过程以迭代的方式来判断, 直到探头两侧接触的差异不区分的眼睛。由于该对准程序依赖于操作者主观视觉检查, 重现性较低。
随后, 开发了一种更客观的方法, 由安装在基板下的力传感器组成, 用来测量表面上探头接触时施加的作用力。12通过调整倾斜角度来实现对齐, 从而使施加的力最大化, 这表明所有探头同时处于接触中。该方法表明, 对0.004°内的表面平行是可能的。这项 "力反馈水准" 现已在两份独立报告中实施为全自动系统。13,14都使用在基板下方或阵列上方安装的力传感器的三重, 并测量探针阵列和曲面之间接触所施加的作用力量。这些系统给出了高精度, 报告≤0.001°的不协调度超过1厘米长,14或≤ 0.0003°over 1.4 厘米.13这些自动对齐系统还提供了大量节省操作员时间和总时间来完成光刻工艺。
这种技术所启用的高通量表面制造的一个主要应用是细胞培养基质的生成。现在已经确定了细胞表型可以通过控制细胞和表面特征之间的初始相互作用来操作, 并且可以在纳米尺度上增强。15具体地说, 扫描探针光刻方法已被证明是一种简便的方法来生产各种 nanofabricated 表面的这种细胞培养实验。16例如, 表面呈现的纳米结构的自组装膜和细胞外基质蛋白模板的 DPN 已被用来研究纳米改性材料在材料诱导分化的潜力干细胞。17
该协议描述了一种改进的原子力显微镜 (AFM) 系统的使用, 它能够实现大面积的 PPL。我们详细介绍了用多力传感器检测力的方法, 作为确定探针表面接触的手段, 以及自动化迭代对准过程的算法。对这些模式的后续功能化与细胞外基质蛋白纤维连接蛋白和人骨髓间充质干细胞 (hMSC) 的培养进行了描述, 作为一种用于细胞培养的人工表面的证明。

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexAFM mounted on a motorised 5-axis (XYZ&#920;&#934;) translation and goniometer stage</td>
			<td>NanoSurf</td>
			<td>P40008</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM control software</td>
			<td>NanoSurf</td>
			<td>C3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Engraving pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z225568</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PlasmaFlo</td>
			<td>Harrick plasma</td>
			<td>PDC-FMG-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Economy Dry Oxygen Service Pump</td>
			<td>Harrick plasma</td>
			<td>PDC-OPE-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>Stuart</td>
			<td>SB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Thermo Fisher Scientific</td>
			<td>5311-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Water Purification System</td>
			<td>Merck Millipore</td>
			<td>ZRXQ015WW</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Humidity Generator</td>
			<td>proUmid</td>
			<td>MHG32</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline Plus Pipette 100-1000 &#181;L</td>
			<td>Sartorius</td>
			<td>728070</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon masters</td>
			<td>NIL Technology</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright snapshot fluorescence microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope objectives</td>
			<td>Olympus</td>
			<td>10x and 60x UPlan FLN &#8734;/-/FN 26.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright bright field microscope</td>
			<td>Leica</td>
			<td>DM 2500M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>Ultrawave Ltd.</td>
			<td>U95</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet for recording and intepreting automated alignment results</td>
			<td>Microsoft</td>
			<td>Excel</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>34863</td>
			<td>FLAMMABLE</td>
		</tr>
		<tr>
			<td>Microscope Sildes, Clear, Ground</td>
			<td>Thermo Fisher Scientific</td>
			<td>451000</td>
			<td></td>
		</tr>
		<tr>
			<td>(7&#8211;8% vinylmethylsiloxane)-dimethylsiloxane copolymer, trimethylsiloxy-terminated</td>
			<td>Gelest</td>
			<td>VDT-731</td>
			<td></td>
		</tr>
		<tr>
			<td>1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane</td>
			<td>Gelest</td>
			<td>SIT7900.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution</td>
			<td>Sigma-Aldrich</td>
			<td>479527</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>(25&#8211;35% methylhydrosiloxane)-dimethylsiloxane copolymer, trimethylsiloxane-terminated</td>
			<td>Gelest</td>
			<td>HMS-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Boat 100 mL</td>
			<td>Scientific Laboratory Supplies</td>
			<td>BALI828</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Appleton Woods</td>
			<td>KS230</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SARSTEDT</td>
			<td>82.1473</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Thermo Fisher Scientific</td>
			<td>ST10-031T</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Carbon Tape</td>
			<td>Agar scientific</td>
			<td>AGG3939</td>
			<td></td>
		</tr>
		<tr>
			<td>16-Mercaptohexadecanoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>448303-1G</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>34852</td>
			<td>FLAMMABLE</td>
		</tr>
		<tr>
			<td>Gold coated microscope slide</td>
			<td>Sigma-Aldrich</td>
			<td>643203</td>
			<td>Once opened gold will remain reactive to thiols for at least 1 month</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Iron(III) nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>529303</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>84415</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>(11-Mercaptoundecyl)hexa(ethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>675105</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(II) nitrate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>203106</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM Mesenchymal Stem Cell Growth Medium</td>
			<td>Lonza UK</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells</td>
			<td>Lonza UK</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Multifuge X1 Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004210</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Centrifuge Tubes</td>
			<td>Greiner Bio-One</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>P/0840/53</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>&#34;Detergent&#34; in manuscript</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-fibronectin antibody</td>
			<td>Abcam</td>
			<td>ab2413</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3912</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>10253041</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 tissue culture flask</td>
			<td>Thermo Fisher Scientific</td>
			<td>10790113</td>
			<td></td>
		</tr>
		<tr>
			<td>cantilever</td>
			<td>BudgetSensor</td>
			<td>ContAl-G</td>
			<td></td>
		</tr>
	</tbody>
</table>

large-area scanning probe nanolithography facilitated by automated alignment and its application to substrate fabrication for cell culture studies

扫描探针显微镜已使创建了各种方法的建设性 ("添加剂") 自上而下制造纳米尺度的特征。从历史上看, 扫描探针光刻的一个主要缺点是单探头系统的固有的低吞吐量。通过使用多个探测器阵列来解决这一问题, 从而提高了光刻吞吐量。为了实现这种并行光刻, 探针阵列与基体表面的精确对准是至关重要的, 因此当光刻模式开始时, 所有探针都能同时与表面接触。该协议描述了聚合物笔光刻在厘米大小的区域内产生纳米尺度特征的利用, 通过使用一种快速、精确和自动对准探针阵列的算法来促进。在这里, 光刻的硫醇的黄金基板显示的特点, 高均匀性的生成。这些模式, 然后功能性与纤连蛋白用于在表面定向细胞形态学研究的背景下使用。

为了检查自动对齐是否成功, 已检查从对齐数据 (在步骤4.8 中的电子表格中) 绘制的图表。如果对齐过程成功, 则两个地块对应于采样阶段沿θ和φ轴倾斜的角度, 显示了一系列上升和下降的数据点。在每个地块中, 两个数据点的线性拟合显示了一个定义良好的相交 "峰值", 表示最大z扩展和实现对齐的相应角度 (图 4A 和 4B)。此过程重复四次 (即每轴两次), 并绘制为一组四坐标。因此, 每对坐标的交集都显示出整体最佳角度 (图 4C)。13在对齐不成功的情况下, 可以观察到, 它们对应的θ和φ角图不提供高质量的线性匹配, 或者不相交 (图 5)。这种失败的对齐通常是由于阵列被不正确地修剪或装入探头持有者 (步骤1.7、1.8 和 2.2)。在这些情况下, 数组被丢弃, 一个新的准备和装载 (步骤1和 2), 并重复对齐过程 (步骤 4)。
成功地对齐和 MHA 后, 用侧向力显微镜对花纹金基片进行成像, 检查是否发生了沉积。对印刷表面进行更大面积的检查时, 还通过基体的光学显微镜对未被沉积的硫醇保护的金进行蚀刻 (图 6和图 7)。但是, 蚀刻图案不能用于进一步的功能化, 只应用于确认一批印刷表面基板的代表性样品的图案。如果蚀刻图案显示与单个钢笔对应的空白区域 (图 8), 则此结果指示探测阵列的生产未成功完成, 并且某些探头已损坏或丢失。探测器的这种不均匀性可能是由于使用了氟涂层已磨损的老主控形状 (步骤 1.3), 导致在阵列与主控形状分离时, 一些探头被撕掉。在这些情况下, 应使用新的主控形状。结果也可能是由于在玻璃衬底和主 (步骤 1.5) 之间存在气泡, 或者在固化后探头阵列与主控形状没有完全分离 (步骤 1.8)。
荧光灯显微图像的纤维连接蛋白功能性表面孵化 hMSCs 也收集 (图 9)。一般情况下, 所有基质在体外培养环境中都是稳定的, 细胞在被隔离的 20 x 20 组特征的情况下附着并适应了印刷图案的形态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56967/56967fig1.jpg"> 图1。聚合物笔光刻的示意图表示,通过探针尖端的水半月板显示分子墨水的传输.(a) 侧面视图和 (B) 聚合物笔阵列的顶部视图表明, 当探头阵列和表面基板完全对准时, 所有探头同时与表面接触, 导致并行光刻。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56967/56967fig2.jpg"> 图2。聚合物笔光刻原理图成立.(A) 扩大了实验装置的侧面视图, 准备好的探头阵列附着在探针支架上, 安装在 AFM 扫描仪上。基板放置在舞台上, 下面是三力传感器的位置。(B) 装配式仪器的表示, 显示 AFM 扫描头相对于样品阶段。(C) 显示力传感器位置的底部视图。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56967/56967fig3.jpg"> 图3。描述对齐过程的光谱学程序的示意图表示法.AFM 扫描仪被设置为在100毫秒内, 在250毫秒的位置进行10µm 的探针, 然后在100毫秒内收缩10µm, 然后在收回的位置为250毫秒举行。然后在整个对准过程中重复该运动。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56967/56967fig4.jpg"> 图4。图示成功对齐的图表.z 位置图对倾斜角度 (A) θ和 (B) φ为一个成功的对齐, 在那里表明测量的实际值和+表明最适合用最小二乘法。(C) φ与θ拟合角度的关系图, 达到最大 z 位置的四点。标记的交点是两个轴的最终最佳倾斜角度。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56967/56967fig5.jpg"> 图5。图示不成功的对齐方式.z 位置图对倾斜角度 (A) θ和 (B) φ为一个不成功的对齐, 在那里指示测量的实际值和+表明最适合用最小二乘法。(C) φ与θ拟合角度的关系图, 达到最大 z 位置的四点。没有明确的最优或相交点, 因此不解决最佳对准角。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56967/56967fig6.jpg"> 图6。说明光学显微学和原子力显微图像的黄金基质, 被排列的 MHA 阵列, 然后蚀刻.(a) 和 (B) 依次放大蚀刻图案的光学显微图像;(C) 是一个单一模式网格的 AFM 地形图像。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56967/56967fig7.jpg"> 图7。MHA 被排列的 PPL 阵列, 然后蚀刻的金色基底的光学显微图像.(a) 和 (B) 依次放大蚀刻图案的光学显微图像, (C) 是一种显示大面积均匀图案的低放大图像。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56967/56967fig8.jpg"> 图8。说明了一种金色基体的光学显微图像, 它的 MHA 不均匀, 然后蚀刻.预定的模式 (显示在嵌入) 是重复网格20点线排列在20行, 每两条线产生的z轴延长1µm (范围从5到-5 µm)。可以看到, 在某些区域, 由于在这些位置中缺少探测器, 因此不会生成任何模式。在仅产生两行点的区域中, 此结果表示探测器存在, 但它与完全运行的探头的高度不相同, 因此仅当阵列扩展到完全z轴距离时才会沉积特征。在这个图像中, 对比度被刻意改变, 以使观察部分印刷区域。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56967/56967fig9.jpg"> 图9。荧光显微图像的 hMSCs 培养的纤维粘连蛋白阵列模板的人.(a) 和 (B) 是显示单个细胞的高放大图像。(C) 在没有附着单元的情况下显示纤连阵列的示例模式, (D) 是在网格排列中培养的单元格的广域图像 (在镶嵌图中也显示了印刷图案的示意图)。细胞染色显示纤维粘连蛋白 (红色), F 肌动蛋白 (绿色) 和细胞核 (蓝色)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56967/56967fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies at JoVE.com

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

在这里, 我们提出了一个广域扫描探针光刻的协议, 由探针阵列的迭代对准所启用, 以及利用光刻模式进行细胞表面相互作用研究。

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

large-area-scanning-probe-nanolithography-facilitated-automated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K 次观看.  University of Manchester. 在这里, 我们提出了一个广域扫描探针光刻的协议, 由探针阵列的迭代对准所启用, 以及利用光刻模式进行细胞表面相互作用研究。

视频: Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

科研

生物工程

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

冲击波应用细胞培养

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

用于小型细胞处理的自动逆流离心系统

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...