This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

<ol>
	<li>Silva, G. 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=Selective+differentiation+of+neural+progenitor+cells+by+high-epitope+density+nanofibers.">Selective differentiation of neural progenitor cells by high-epitope density nanofibers.</a> Science. 303 (5662), 1352-1355 (2004).</li><li>Yim, E. K. F., Pang, S. W., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Nanostructures+Inducing+Differentiation+of+Human+Mesenchymal+Stem+Cells+into+Neuronal+Lineage.">Synthetic Nanostructures Inducing Differentiation of Human Mesenchymal Stem Cells into Neuronal Lineage.</a> Exp. Cell Res. 313 (9), 1820-1829 (2007).</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 (12), 997-1003 (2007).</li><li>Oh, S., 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=Stem+Cell+Fate+Dictated+Solely+by+Altered+Nanotube+Dimension.">Stem Cell Fate Dictated Solely by Altered Nanotube Dimension.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (7), 2130-2135 (2009).</li><li>Brunetti, V., 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=Neurons+Sense+Nanoscale+Roughness+with+Nanometer+Sensitivity.">Neurons Sense Nanoscale Roughness with Nanometer Sensitivity.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (14), 6264-6269 (2010).</li><li>McMurray, 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=Nanoscale+Surfaces+for+the+Long-term+Maintenance+of+Mesenchymal+Stem+Cell+Phenotype+and+Multipotency.">Nanoscale Surfaces for the Long-term Maintenance of Mesenchymal Stem Cell Phenotype and Multipotency.</a> Nat. Mater. 10 (8), 637-644 (2011).</li><li>Yim, E. K. 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=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Gerecht, S., 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+Effect+of+Actin+Disrupting+Agents+on+Contact+Guidance+of+Human+Embryonic+Stem+Cells.">The Effect of Actin Disrupting Agents on Contact Guidance of Human Embryonic Stem Cells.</a> Biomaterials. 28 (28), 4068-4077 (2007).</li><li>Bettinger, C. J., Zhang, Z., Gerecht, S., Borenstein, J. T., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+in+vitro+Capillary+Tube+Formation+by+Substrate+Nanotopography.">Enhancement of in vitro Capillary Tube Formation by Substrate Nanotopography.</a> Adv. Mater. 20 (1), 99-103 (2008).</li><li>Thakar, R. G., Ho, F., Huang, N. F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Commun. 307 (4), 883-890 (2003).</li><li>Lee, M. 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=Direct+differentiation+of+human+embryonic+stem+cells+into+selective+neurons+on+nanoscale+ridge/groove+pattern+arrays.">Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays.</a> Biomaterials. 31 (15), 4360-4366 (2010).</li><li>Moe, A. A. 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=Microarray+with+micro-+and+nano-topographies+enables+identification+of+the+optimal+topography+for+directing+the+differentiation+of+primary+murine+neural+progenitor+cells.">Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.</a> Small. 8 (19), 3050-3061 (2012).</li><li>Dang, J. M., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+induction+of+aligned+mesenchymal+stem+cell+sheets+by+culture+on+thermally+responsive+electrospun+nanofibers.">Myogenic induction of aligned mesenchymal stem cell sheets by culture on thermally responsive electrospun nanofibers.</a> Adv. Mater. 19 (19), 2775-2779 (2007).</li><li>Dasgupta, N., 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=Thermal+co-reduction+approach+to+vary+size+of+silver+nanoparticle:+its+microbial+and+cellular+toxicology.">Thermal co-reduction approach to vary size of silver nanoparticle: its microbial and cellular toxicology.</a> Environ. Sci. Pollut. Res. 23 (5), 4149-4163 (2016).</li><li>Ranjan, S., 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=Microwave-irradiation-assisted+hybrid+chemical+approach+for+titanium+dioxide+nanoparticle+synthesis:+microbial+and+cytotoxicological+evaluation.">Microwave-irradiation-assisted hybrid chemical approach for titanium dioxide nanoparticle synthesis: microbial and cytotoxicological evaluation.</a> Environ. Sci. Pollut. Res. 23 (12), 12287-12302 (2016).</li><li>Deckman, H. W., Dunsmuir, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+lithography.">Natural lithography.</a> Appl. Phys. Lett. 41 (4), 377-379 (1982).</li><li>Dalby, M. J., Riehle, M. O., Johnstone, H., Affrossman, S., Curtis, A. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Reaction+of+Endothelial+Cells+to+Polymer+Demixed+Nanotopography.">In vitro Reaction of Endothelial Cells to Polymer Demixed Nanotopography.</a> Biomaterials. 23 (14), 2945-2954 (2002).</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 (4), 1171-1196 (2005).</li><li>Yin, Y., Lu, Y., Gates, B., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-Assisted+Self-Assembly:+A+Practical+Route+to+Complex+Aggregates+of+Monodispersed+Colloids+with+Well-Defined+Sizes,+Shapes,+and+Structures.">Template-Assisted Self-Assembly: A Practical Route to Complex Aggregates of Monodispersed Colloids with Well-Defined Sizes, Shapes, and Structures.</a> J. Am. Chem. Soc. 123 (36), 8718-8729 (2001).</li><li>Tada, 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=Directed+Self-Assembly+of+Diblock+Copolymer+Thin+Films+on+Chemically-Patterned+Substrates+for+Defect-Free+Nano-Patterning.">Directed Self-Assembly of Diblock Copolymer Thin Films on Chemically-Patterned Substrates for Defect-Free Nano-Patterning.</a> Macromolecules. 41 (23), 9267-9276 (2008).</li><li>Cheng, J. Y., Rettner, C. T., Sanders, D. P., Kim, H. C., Hinsberg, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+self-assembly+on+sparse+chemical+patterns:+rectifying+and+multiplying+lithographic+patterns+using+block+copolymers.">Dense self-assembly on sparse chemical patterns: rectifying and multiplying lithographic patterns using block copolymers.</a> Adv. Mater. 20 (16), 3155-3158 (2008).</li><li>Colburn, 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=Step+and+flash+imprint+lithography:+a+new+approach+to+high-resolution+patterning.">Step and flash imprint lithography: a new approach to high-resolution patterning.</a> Proc. SPIE. 3676 ((Pt. 1, Emerging Lithographic Technologies III)), 379-389 (1999).</li><li>Ahn, S. H., Guo, 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=High-speed+roll-to-roll+nanoimprint+lithography+on+flexible+plastic+substrates.">High-speed roll-to-roll nanoimprint lithography on flexible plastic substrates.</a> Adv. Mater. 20 (11), 2044-2049 (2008).</li><li>Vieu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+beam+lithography:+resolution+limits+and+applications.">Electron beam lithography: resolution limits and applications.</a> Appl. Surf. Sci. 164, 111-117 (2000).</li><li>Nagase, T., Gamo, K., Kubota, T., Mashiko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching.">Direct fabrication of nano-gap electrodes by focused ion beam etching.</a> Thin Solid Films. 499 (1-2), 279-284 (2006).</li><li>Juodkazis, S., 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=Two-photon+lithography+of+nanorods+in+SU-8+photoresist.">Two-photon lithography of nanorods in SU-8 photoresist.</a> Nanotechnology. 16 (6), 846 (2005).</li><li>Yang, Y., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surfacing+for+regenerative+medicine.">Nanoscale surfacing for regenerative medicine.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5), 478-495 (2010).</li><li>Yang, Y., Kulangara, K., Sia, J., Wang, L., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features.">Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features.</a> Lab Chip. 11 (9), 1638-1646 (2011).</li><li>Wang, 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=Nanotopographical+modulation+of+cell+function+through+nuclear+deformation.">Nanotopographical modulation of cell function through nuclear deformation.</a> Acs Appl. Mater. Inter. 8 (8), 5082-5092 (2016).</li><li>Lee, J. N., Park, C., 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=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75 (23), 6544-6554 (2003).</li><li>Yang, Y., Kulangara, K., Lam, R. T. S., Dharmawan, R., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior.">Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior.</a> ACS Nano. 6 (10), 8591-8598 (2012).</li><li>Hua, 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+Imprint+Lithography+with+Molecular-Scale+Resolution.">Polymer Imprint Lithography with Molecular-Scale Resolution.</a> Nano Lett. 4 (12), 2467-2471 (2004).</li><li>Delamarche, E., Schmid, H., Michel, B., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+molded+polydimethylsiloxane+microstructures.">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li></ol>

The authors have nothing to disclose.

نقدم طريقة بسيطة، وبأسعار معقولة، ولكن تنوعا لتوليد مساحة كبيرة من الركيزة nanopatterned. لتوسيع بأمانة nanopatterns محددة للغاية، وينبغي إيلاء اهتمام كبير لعدة خطوات حاسمة. أول واحد هو تقليم قوالب PDMS متعددة. يجب إزالة المناطق Unpatterned من القوالب PDMS. بالإضافة إلى ذلك، الجدران الجانبية من القوالب يجب أن تقطع عموديا المثالي قدر الإمكان لتقليل الفجوات بين القوالب. بشكل جماعي، وجزء من مناطق unpatterned في قالب غرزة النهائي يمكن تخفيض. ثانيا، على سطح nanopatterned من هذه القوالب PDMS يحتاج إلى الانحياز من دون أي تحريف في الركيزة السيليكون. لأن النانو PDMS عرضة للتشوه، فمن الأهمية بمكان لوضع الأسطح nanopatterned ضد الجانب مرآة الركيزة السيليكون بلطف وبشكل متساو (تجنب احتباس الهواء بين القالب PDMS وسطح السيليكون). سيتم محاذاة قوالب PDMS في أقرب وقت ممكن، ولكن ليس لمس نيىghboring قوالب لمزيد من تقليل الجزء unpatterned من العفن غرزة النهائي. وإلا فإن النانو تطرق تشوه خلال nanoimprinting. ثالثا، سمك قوالب PDMS قد تختلف من دفعة لدفعة، وبالتالي فمن الأهمية بمكان لجعل الزي سمك بالإضافة إلى جعل سمك كل قالب موحد من التسوية القالب سي صغير تماما قبل PDMS الصب. وعلى الرغم من الاختلاف في سماكة عبر قوالب PDMS يمكن تعويضه عن طريق ضبط سمك PDMS prepolymer (لاصق) طبقة يلقي على شريحة زجاجية، يمكن أن طبقة سميكة prepolymer يكون مشكلة. قد يتم سحب prepolymer من خلال الفجوات بين القوالب PDMS إلى السطح المزخرف من قبل القوة الشعرية، وبالتالي تلف nanopatterns. تباين سمك يمكن التقليل من خلال إعداد نفس الكمية من PDMS خليط عندما صب من العفن EBL. ونتيجة لذلك، رقيقة PDMS طبقة prepolymer يمكن استخدامها. بدلا من ذلك، وعلاج جزئيا prepolymeوطبقة ص زيادة اللزوجة، وبالتالي الحد من زيادة وفي النهاية القضاء على الضرر المحتمل من الأسطح nanopatterned. تقنية غرزة محدودة بسبب طبيعة المرنة من PDMS. على الرغم من الطباعة الحجرية الناعمة تم تطبيقه على تكرار أحجام ميزة صغيرة مثل 2 نانومتر 32 و، من حيث المبدأ، يمكن أن تصل إلى دقة أقل من 0.5 نانومتر 18، وملامح PDMS النانو لا يمكن تكرارها لا تشوبه شائبة عند الجانب نسبة الارتفاع إلى العرض هي أيضا عالية (&gt; 2) أو منخفضة جدا (&lt;0.2). قد تنهار في nanofeatures عند نسبة الارتفاع مرتفعة جدا، أو يؤدي إلى عدم كفاية الإغاثة عند استخدام الطوابع PDMS من &lt;0.2 نسبة 33. وعلاوة على ذلك، وقوالب متعددة PDMS لا يمكن مخيط بسلاسة بسبب الفجوات والتشذيب ناقصة من القوالب PDMS، وبالتالي هناك مناطق unpatterned والمنحرفة (ولا سيما بالنسبة للnanopatterns المستمر مثل nanogratings). ونظرا لنسبة صغيرة من انشقمساحة تزيد عن المساحة الإجمالية، وتقنية غرزة لا يزال يوفر وسيلة بسيطة ومعقولة التكلفة لإنتاج مساحة واسعة من ركائز nanopatterned. وبالإضافة إلى ذلك، عندما يتم nanoimprinted القالب مخيط إلى الركيزة البوليمر، قد تتدفق البوليمر المنصهر في فرجة، مما أدى إلى استواء السطح (الشكل 1A). سطح متفاوتة يجعل صعوبة في جمع عينات للتحاليل البيولوجية الخلوية أو الجزيئية. في تطبيقات ميكروفلويديك، يتسبب في زيادة أيضا ختم ناقصة عندما مختومة microchannels ضد الركيزة المزخرفة. يمكن بسهولة أن تحل المشكلة سطح غير مستو عن طريق تطبيق البوليمر تقنية رقيقة للحد من الزيادة من خلال ضبط سماكة الفيلم (الشكل 1B). على الرغم من أن تقنية غرزة يحتاج إلى القالب الرئيسي المحدد للتوسع، فهي بسيطة وبأسعار معقولة مقارنة مع التقنيات الأخرى مثل الطباعة الحجرية خطوة وفلاش وnanoimprinting lithogra لفة إلى لفةفيز. تتطلب تقنية غرزة فقط وحات الساخنة وسيلة لممارسة الضغط القوات أثناء عمليات خياطة وnanoimprinting، ولكن ليس معدات باهظة الثمن. وعلاوة على ذلك، فإن عملية غرزة يمكن أن تجرى في بيئة نظيفة، ولكن ليس بالضرورة في غرف الأبحاث. تقنية غرزة هو أيضا تنوعا. وبالإضافة إلى توسيع وnanopattern مماثلة لمساحة كبيرة، ويمكن تطبيق هذه التقنية غرزة لقوالب تتكون من الجزئي و / أو النانو ملامح من مختلف الأشكال والأبعاد والترتيبات. في هذا الصدد، مكتبة توفيقية من الصغرى / nanotopographies يمكن أن يبنى على توفير منصة عالية الإنتاجية للتحقيق في التفاعلات خلية التضاريس. من المحتمل أن يتم تمديد هذه التقنية غرزة بسيطة ومعقولة التكلفة وتنوعا في ابتكار أجهزة متناهية الصغر / النانو مع المكونات الهجينة.

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table><tbody><tr><td>JEOL field emission SEM</td><td>JEOL</td><td>JSM-7600F</td><td>EBL</td></tr><tr><td>E-beam evaporator</td><td>Kurt J. Lesker</td><td>Model: LAB 18 e-beam evaporator</td><td>nickel deposition</td></tr><tr><td>Trion Minilock III ICP/RIE</td><td>Trion technology</td><td>Model: Minilock-phantom III</td><td></td></tr><tr><td>Press machine</td><td>PHI Hydraulic Press</td><td>Molde: SQ-230H</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>Modle: WS-400A-6NPP-LITE</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; critical dryer</td><td>Tousimis</td><td>Modle: Autosamdri-815</td><td></td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>1080</td><td></td></tr><tr><td>Aluminum plates</td><td>McMaster-carr</td><td>9057K123</td><td></td></tr><tr><td>Teflon sheets</td><td>McMaster-carr</td><td>8711K92</td><td></td></tr><tr><td>100 mm Petri dish</td><td>FALCON</td><td>353003</td><td></td></tr><tr><td>60 mm Petri dish</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Glass slide</td><td>Fisher Scientific</td><td>12-550-34</td><td></td></tr><tr><td>Disposable weighing boats</td><td>Fisher Scientific</td><td>13-735-743</td><td></td></tr><tr><td>Glass desiccator</td><td>Fisher Scientific</td><td>02-913-360</td><td></td></tr><tr><td>Plastic desiccator</td><td>Bel-Art Products</td><td>F42025-000</td><td></td></tr><tr><td>Hotplate</td><td>Fisher Scientific</td><td>1110049SH</td><td></td></tr><tr><td>Tweezer</td><td>Ted Pella, inc.</td><td>5726</td><td></td></tr><tr><td>Blade</td><td>Fisher Scientific</td><td>S17302</td><td></td></tr><tr><td>Metal blocks</td><td>McMaster-carr</td><td></td><td></td></tr><tr><td>Punch</td><td>Brettuns Village Leather Craft Supplies</td><td>Arch punch</td><td></td></tr><tr><td>Poly(methyl methacrylate)</td><td>MicroChem</td><td>495 PMMA A4</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td></td></tr><tr><td>Polystyrene</td><td>Dow Chemical</td><td>Styron 685D</td><td></td></tr><tr><td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td><td>Oakwood Chemical</td><td>7142</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>MIBK/IPA at 1: 3 ratio</td><td></td></tr><tr><td>Remover</td><td>MicroChem</td><td>Remover PG</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Toluene</td><td>Fisher Scientific</td><td>T324-500</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle medium</td><td>Sigma Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microsopy Science</td><td>15712-S</td><td></td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>Fisher Chemical</td><td>G151-1</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL-185</td><td></td></tr></tbody></table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

<html> تقنية غرزة يمكن أن يولد مساحة واسعة من ركائز nanopatterned مع الدقة العالية. الشكل 1A 1B وعرض مساحة كبيرة من nanopatterns نقلها من العفن PDMS مخيط لوحة PS و PS طبقة رقيقة على ركيزة سي، على التوالي. المقارنة بين الأصلي والعفن مكتوبة EBL (الشكل 1C) وPDMS العمل النهائية الركيزة (1D الشكل) تؤكد أن nanopatterns مكتوبة EBL-يمكن نقلها بأمانة إلى الركيزة العمل. Nanotopography من مختلف الهندسة والأبعاد يمكن استخدامها لتعديل سلوك الخلية. كما هو موضح في الشكل رقم 2 مع A549، وهي القاعدية خط الخلايا الظهارية adenocarcinomic كخلايا نموذج، يمكن للnanogratings متباين الخواص استطال الخلايا على طول اتجاه nanograting مقارنة مع التشكل المتعدد الأقطاب الذي تعرض خلايا A549 على عمود نانوي الخواص. الإقليم الشمالي &quot;FO: المحافظة على together.within الصفحات =&quot; 1 &quot;&gt; <img alt="شكل 1" src="/files/ftp_upload/54840/54840fig1.jpg" /> الشكل 1. جيل من مساحة واسعة من ركائز nanopatterned باستخدام تقنية غرزة. (أ، ب) الصور الضوئية للnanopatterns نقلها إلى لوحة PS و PS رقيقة، على التوالي. تشير الأسهم إلى رفع البوليمر في الفجوات من القوالب PDMS مخيط. (ج، د) الصور SEM من nanopatterns في قالب EBL وPDMS النهائية الركيزة العمل، على التوالي. القضبان على نطاق وهي 1 ميكرون. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/54840/54840fig2.jpg" /> الشكل 2. Nanotopographical التضمين خلية انتشار خلايا A549. </strong&gt; خلايا A549 نمت على (أ) nanogratings من 500 نانومتر في عرض الخط، 500 نانومتر في تباعد و 560 نانومتر في الطول و (ب) عمود نانوي من 500 نانومتر في القطر، 450 نانومتر في تباعد من الحافة إلى الحافة و 560 نانومتر في ارتفاع ، على التوالي. القضبان على نطاق وهي 10 ميكرون. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

توسيع Nanopatterned ركائز باستخدام تقنية غرزة لNanotopographical التحوير سلوك الخلية

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.5K Views.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Video: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

الاستنسل Micropatterning من الخلايا الجذعية متعددة القدرات البشرية لجس التنظيم المكاني للتمايز الأقدار

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Developmental Biology

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

الشعرية قوة الطباعة الحجرية لهندسة الأنسجة القلبية

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

التصنيع الدقيق لأنماط ذهب نانوية مسامية للدراسات التفاعل الخلوي المواد

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

الزخرفة الخلية على Photolithographically معرفة Parylene-C: شافي<sub&gt; 2</sub&gt; ركائز

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

تمتد خلايا Micropatterned على غشاء PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

بسيطة خالية من الطباعة الحجرية خلية واحدة Micropatterning باستخدام الليزر قطع الإستنسل

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

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.

منهج تنوعا من الزخرفة البروتينات والخلايا

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

تصنيع نانوباتيرن التدرج الحراري نانويمبرينتينج تقنية والفحص لاستجابة الخلايا البشرية تشكيل مستعمرة غشائي

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

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

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

السيطرة على هندسة الخلية من خلال الأشعة تحت الحمراء الليزر بمساعدة Micropatterning

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

Pattern Generation for Micropattern Traction Microscopy

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

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

توليد النمط لمجهر الجر المجهري

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