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.

Bir EBL Kalıp PDMS Kalıpları 1. çoğaltma <ol><li> 29 Silikon kalıp fabricate <ol><li> Spin kat 200 ul polimetil metakrilat (PMMA), 1 dakika boyunca 2500 rpm&#39;de 1 x 1 cm, silikon (Si) alt tabaka üzerinde çözelti ince bir film meydana getirmek üzere. </li><li> 2 dakika boyunca 180 ° C&#39;de Si alt-tabaka üzerinde PMMA filmi fırında. </li><li> 300 MCU / cm2 bir alan dozunda odaklanmış bir elektron ışını ile PMMA film tasarlanmıştır nanopattern yaz. </li><li> 80 saniye geliştirici PMMA nanopattern geliştirin. </li><li> 10 kV, 0.5 mA emisyon akımı ve 0.5 a / sn&#39;lik bir depozisyon oranının bir çıkış voltajı bir E-ışın demeti buharlaştırıcı kullanılarak kalınlığı 50 nm bir nikel tabaka ile PMMA nanopattern bırakın. </li><li> 20 dakika boyunca, 80 ° C&#39;de 20 mi çıkarıcı PMMA parçası kaldırın. </li><li> Reaktif iyon aşındırma (RIE) Si substrat içine nanopattern istenen derinlik Si kalıp almak için. NOT: tetraklorohidreks Gaz karışımıafluoromethane (KF 4), 150 W, 400 W ve RIE bir güç indüktif olarak birleştirilmiş plasma (ICP) güçte / oksijen (O2) (% 90 /% 10), 560 nm&#39;lik bir derinliğe kadar Si alt-tabakanın etch için kullanılır. </li></ol></li><li> Silanize Si kalıp <ol><li> 100 mm PS Petri kabı bir cam lamel Si kalıp koyun ve bir çeker ocak içinde bulunan cam bir kurutucuda aktarın. </li><li> lamel 10 ul 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane bırakın. Dikkat: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane cilt korozyon ve ciddi göz hasarına neden olabilir. Uygun kişisel koruyucu ekipman (KKE) giyin. </li><li> Kısmen Petri kabı örtün. </li><li> Si kalıp silanizasyondan tamamlamak için bir duman başlığı içinde, 5 saat boyunca vakum altında desikatörde tutun. </li></ol></li><li> PDMS ön-polimerin hazırlanması <ol><li> tek kullanımlık bir tartım kabı 10 gr PDMS reçine ve 1.05 g sertleştirme ajanı tartılır. </li><li> Bir plastik kaşık kullanarak iyice PDMS prepolimeri karıştırın. </li><li> daha berrak bir karışım görülmektedir kadar yaklaşık 20 dakika boyunca vakum altında plastik bir desikatör içinde PDMS prepolimer gaz çıkışına. </li></ol></li><li> PDMS kalıpları çoğaltmak <ol><li> 60 mm Petri kabındaki Silanlanmış Si kalıp koyun. </li><li> Petri kabındaki Si kalıp üzerine PDMS prepolimeri dökün. </li><li> tüm kabarcıklar kaybolur dek yaklaşık 10 dakika süreyle bir plastik desikatörde ve gazını Petri kabı yerleştirin. </li><li> Bir ocak petri aktarın ve 4 saat 70 ° C&#39;de PDMS prepolimeri tedavi. </li><li> dikkatle cımbız kullanarak Si kalıp PDMS kalıp soyulabilir. Not: PDMS kalıplar bir haftaya kadar çevre şartlarında muhafaza edilebilir. Kür sonra, PDMS kalıplar 30 bazı çapraz bağlanmamış PDMS reçine molekülleri ve artık sertleştirme ajanı vardır. düşük molekül ağırlıklı moleküller yavaş yavaş nüfuz ve zaman içinde yüzeyde birikebilir. Bu PDMS yüzeyi 31 topografik ve mekanik özelliklerini etkiler. difFüzyon bir hafta içinde çok önemli değildir. </li></ol></li></ol> Büyük Bir, Tek Kalıp içine PDMS Kalıpları 2. Dikiş <ol><li> Adım 1.4 tekrarlayarak birden PDMS kalıplar hazırlayın. NOT: PDMS karışımın aynı miktarda aynı kalınlıkta PDMS kalıpları elde etmek için her zaman tartın. </li><li> Böyle bir optik mikroskop altında nanogratings olarak anizotropik PDMS nanopatterns yönünü belirlemek ve bir marker kalem ile PDMS kalıp ters işaretleyin. NOT: Bu nanopillars izotrop nanotopography yönünü işaretlemek için gerekli değildir. </li><li> Davlumbaz etanol ile Si substratı temizlemek ve basınçlı hava ile kurutun. </li><li> Bir bıçak ile her PDMS kalıp desensiz alanları keserek. NOT: dikişli kalıp çevresine alınacaktır PDMS kalıplar için, başkaları ile temas halinde sadece desensiz alanlar kapalı kesilmiş olmalıdır. </li><li> üzerinde nanopattern yüzü aşağı ile kesilmiş PDMS kalıp yerleştirinAyna Si substrat yan ve daha sonra yakın ama çevredeki kalıp (ler) temas etmeyen diğer kalıpları hizalayın. </li><li> PDMS yapışkan tabakanın hazırlanması <ol><li> 0.5 mm kalınlıkta bir tabaka oluşturmak için temiz bir cam slayt (2.5 cm x 7.5 cm), 1 g PDMS prepolimer (1.05: 10: PDMS reçine ve sertleştirici oranı) karışımının gazı alınır Dökme. </li><li> 3-5 dakika boyunca bir sıcak plaka üzerinde 100 ° C&#39;de PDMS tabakanın fırında. katmanı dokunun ve katman kısmen değil tamamen tedavi olduğundan emin olmak için bir iğne kullanın. NOT: Kısmen tedavi PDMS sertleşmemiş PDMS prepolimer gibi akamaz, ancak tedavi PDMS ile karşılaştırıldığında yapışkan. </li></ol></li><li> hizalanmış PDMS kalıp ters PDMS katmanı yerleştirin ve hızlı bir şekilde bu derleme ters ve ocak transfer. </li><li> PDMS yapışkan tabaka ve PDMS kalıpların ters arasında iyi bir temas sağlamak, ve 1 saat 100 ° C&#39;de PDMS yapışkan tabakanın tedavi tertibatının üst kısmında bir metal bloğu kullanarak sıkıştırma kuvveti (5 kPa) uygulanır. &lt;br /&gt; Not: dikkatlice montaj eğimini önlemek için, metal bloğun konumunu ayarlamak. </li><li> Metal bloğunu çıkarın ve Si alt tabakadan tek dikişli PDMS kalıp soyulabilir. </li></ol> PS Yüzeyler üzerinde master Kalıp 3. Nesil Not: cam slayt üzerinde immobilize dikişli PDMS kalıp PS plaka ya da çalışma nano substratlar üretilebilir olan PS ince film, bir ana kalıp oluşturmak için de kullanılabilir. <ol><li> PS plaka üzerinde bir ana kalıp oluşturmak <ol><li> PS tabak hazırlayın <ol><li> İki gün süre ile, 80 ° C&#39;de vakumlu bir fırında PS pelet kurutulur. </li><li> 230 ° C&#39;de, bir pres makinesi önceden ısıtın. </li><li> alttan üste doğru sırayla bir alüminyum plaka, bir politetrafloroetilen (PTFE) levha ve alüminyum ayırıcı birleştirin. </li><li> 3 cm (L) x 3 cm (W) x 0.3 cm (H) bir kare açılışı ile alüminyum ayırıcı yük 3.5 g PS pelet. NOT: boşluk yaklaşık 0&#39;dır.1 PDMS kalıp daha kalın cm ve bu yüzden son nano PS alt-tabaka, yaklaşık 0.1 cm kalınlığındadır. </li><li> Alüminyum ayırıcı başka PTFE levha ve sonra başka bir alüminyum plaka koyun. </li><li> pres makinesinde aksamını yerleştirin. </li><li> 30 dakika boyunca 230 ° C &#39;de PS pelet ısıtın. </li><li> 5 dakika montaj üzerinde bir sıkıştırma basıncı (0,1 Mpa) uygulayın. </li><li> basıncı bırakın ve daha sonra montaj 0.5 MPa basınç basınç yeniden uygulayın. </li><li> 1.5 MPa istenen basınç kadar 0.5 MPa&#39;lık bir basınç artışı ile tekrarlayın Adım 3.1.1.9 ulaşılır. </li><li> pres makinesinin ısıtıcı kapatın ve 1.5 MPa&#39;lık bir sabit basınçta 70 ° C&#39;nin altında soğumasını. </li><li> takımını çıkarın ve PS levha yeniden nem girmesini önlemek için 80 ° C&#39;de vakumlu bir fırında PS plaka saklayın. </li></ol></li><li> PS levha haline Nanoimprint dikişli PDMS kalıp <ol><li> bir alüminyum aralama PS plaka koyun3-inç Si gofret ayarlanır. Not: ara parçasının iç boyutları PS levha ile aynıdır, böylece PS levha doğru aralama uyar. </li><li> 30 dakika boyunca 250 ° C&#39;de bir sıcak plaka üzerinde PS levha ısıtın. </li><li> Erimiş PS plaka üzerinde nanopatterns yüzü aşağı dikişli PDMS kalıp koyun. Not: PDMS kalıp bir tarafı birinci PS levha yüzeyi ile temas halinde yerleştirilir ve başka bir yan arayüzü, hava kabarcıklarının oluşumunu önlemek için PS yüzey ile temas halinde yavaş yavaş indirilir. Dikkat: ocak yüzeyi sıcak. nanoimprinting işlemi sırasında thermogloves kullanın. </li><li> dikişli PDMS kalıp cam slayt bir alüminyum plaka koyun. </li><li> Alüminyum levha metal bloklar kullanılarak bir sıkıştırma basıncı (12.5 kPa) uygulayın ve 3 dakika boyunca bekleyin. NOT: alüminyum levha eğik olmadığından emin olun. </li><li> Kaldırın ve alüminyum plaka metal bloğunu değiştirin ve ben25 kPa basınç basınç arttırın. </li><li> basınçla Adımı tekrarlayın 3.1.2.6 kPa 50 arttı. Not: Bu adım PDMS kalıp ve PS levha arasında sıkışan havayı kaldırmaktır. </li><li> 15 dakika boyunca 50 kPa tutarlı basınç altında 240 ile 250 ° C arasında sıcak plaka sıcaklığı muhafaza edin. </li><li> ocak gözünü kapatın ve tüm kurulum soğumasını. NOT: Bir fan soğutma sürecini hızlandırmak için kullanılabilir. </li><li> PS levha dikişli PDMS kalıp kalkmasına dikkatle sıcaklığı 50 ° C&#39;nin altında sonra metal blokları kaldırmak ve. NOT: PS alt-tabaka ters nanopatterns sahiptir ve çalışma PDMS tabakaları üretmek için bir mastar kalıp olarak kullanılabilir. </li></ol></li></ol></li><li> PS ince film üzerinde bir ana kalıp oluşturmak <ol><li> PS ince film hazırlayın <ol><li> Bir çeker ocak içinde 10 mi toluen içinde 1 g PS çözülür. Dikkat: Toluen Cilt İrritatio neden olabilirn ve ciddi göz hasarı ve uzun süre veya tekrarlanan maruz kalmalar organlara zarar verebilir. Uygun PPE giyin. </li><li> 1 dakika için 2500 rpm&#39;de bir gofret 2-in spin-kaplama 1 mi PS solüsyon 1 mikron kalınlığında PS ince bir film meydana getirmek üzere. </li><li> 3 gün boyunca bir davlumbaz Si gofret PS filmi ayarlayarak film toluen buharlaşır. </li><li> gece boyunca 80 ° C&#39;de bir vakum fırın içinde PS ince film tavlanması. </li></ol></li><li> Nanoimprint PS ince film üzerinde PDMS kalıp <ol><li> bir ocak yer almaktadır PS ince film üzerinde nanotopography yüzü aşağı dikişli PDMS kalıp koyun. </li><li> PDMS kalıp cam tarafındaki metal blokları kullanarak PDMS kalıp 12 kPa basınç basınç uygulayın. </li><li> 180 ° C sıcak plaka sıcaklığı artırmak ve 15 dakika için muhafaza. Dikkat: Erimiş PS filmi yağlayıcı olarak işlev görebilir. kaymasını metal blokları önlemek için dikkat edin. </li><li> ocak gözünü kapatınve tüm kurulum soğumasını. NOT: Bir fan soğutma sürecini hızlandırmak için kullanılabilir. </li><li> PS filmden dikişli PDMS kalıp kalkmasına dikkatle sıcaklığı 50 ° C&#39;nin altına düştüğünde sonra metal blokları kaldırmak ve. NOT: nano PS filmi çalışma PDMS yüzeyler üretmek için bir ana kalıp olarak görev yapacak. </li></ol></li></ol></li></ol> Hücre Davranış 4. Nanotopographical modülasyonu Not: temsilci nanotopographies hücre yayma nanotopographical modülasyonu göstermek için İnsan epitel hücreleri kültür. <ol><li> Uygulamaya bağlı olarak Adım 3.1 veya 3.2 birinden oluşturulan master kalıp PDMS çalışma substratları Cast. </li><li> Içi boş bir çelik kemer zımbası kullanılarak, belirli bir çok-çukurlu plaka (örneğin, 24-çukurlu plaka) konfigürasyonunu uyacak diskler içine nano PDMS substratlar kesti. </li><li> o kuyuların içine PDMS diskleri yerleştirmek için cımbız kullanınFA çok-yuvalı plaka. </li><li> % 70 etanol ve UV ışınlarına maruz kalma, 30 dakika boyunca, her kullanarak PDMS substratlar sterilize edin. </li><li> 1x steril fosfat tamponlu tuzlu su (PBS) ile üç kez PDMS substratlar yıkayın. </li><li> Kat, oda sıcaklığında 30 dakika boyunca hücre dışı matris proteini (yani, 20 ug / ml fibronektin) PDMS alt-tabakalar. </li><li> 5 dakika boyunca PDMS alt tabakaları steril PBS ile üç kez, her durulama. </li><li> % 10 cenin sığır serumu ile Dulbecco tadil edilmiş Eagle ortamı içinde, insan akciğer kanseri hücre A549 askıya alma ve hemasitometre kullanarak hücreleri sayın. </li><li> Bir gün için% 5 CO2 içeren nemli bir atmosferde 37 ° C &#39;de PDMS substratlar ve kültür bunları 2,000 hücre / cm2 bir tohumlama yoğunluğunda hücreler plaka. </li><li> PBS ile üç kez hücreleri yıkayın. </li><li> 4 saat süreyle PBS içinde% 4 paraformaldehid ve% 2 gluteraldehit oluşan bir karışım içinde hücreleri saptamak ve bir CO2 kritik po kullanarak hücrelerin suyunuElektronik mikroskobik gözlem 29 tarama için int kurutma makinası. Dikkat: Paraformaldehyde ve glutaraldehit Ciddi derecede deri yanıkları ve göz hasarına neden olabilir. Bir kimyasal kaput İşlet ve uygun KKE giyerler. </li></ol>

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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.

Biz nano substratın geniş bir alana oluşturmak için basit, uygun fiyatlı, çok yönlü yöntem mevcut. sadakatle yüksek tanımlı nanopatterns genişletmek için, büyük ilgi birçok kritik adımlar dikkat edilmelidir. İlki birden PDMS kalıp Döşeme etmektir. PDMS kalıp desensiz alanlar kaldırılması gerekir. Ayrıca, kalıp yan duvarları kalıplar arasındaki boşlukları en aza indirmek için mümkün olduğunca dikey olarak mükemmel kesilmelidir. Toplu olarak, son dikiş kalıp içinde desensiz alanlar...

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> dikiş tekniği yüksek sadakat ile nano yüzeylerde geniş bir alanda üretebilir. Şekil 1a ve sırasıyla Si substrat PS plaka ve PS ince film dikişli PDMS kalıp aktarılan nanopatterns büyük alanı görüntülemek 1b. Orijinal EBL yazılı kalıbın (Şekil 1c) ve alt tabaka (Şekil 1d) çalışma nihai PDMS arasında karşılaştırma EBL yazılmış nanopatterns sadakatle çalışan alt tabakaya transfer ...

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.

Hücre Davranış Nanotopographical Modülasyon için nano Substratları kullanma Dikiş Tekniği Genişleyen

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

Research

JoVE Journal

Bioengineering

6.5K Görüntüleme.  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.

Farklılaşma Fates Mekansal Organizasyonu Probing İnsan pluripotent kök hücrelerin Stencil micropatterning

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

Gelişim Biyolojisi

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.

Kardiyak Doku Mühendisliği kılcal Kuvvetleri Litografi

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

Biyomühendislik

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.

Hücre-malzeme Etkileşim Araştırmalar Nanogözenekli Altın Patterns mikroimalat

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.

Photolithographically Tanımlı Parylene-C hücre Desenlendirme: SiO<sub&gt; 2</sub&gt; Yüzeyleri

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.

PDMS Zarında Micropatterned Hücreler germe

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.

Lazer Kesim Şablonlar Kullanılarak Basit Litografisiz Tek Hücreli Mikrodesenleme

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.

Desenlendirme Proteinlerin ve Hücreleri Çok Yönlü Yöntemi

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.

Termal Nanoimprinting teknik ve insan endotel hücreleri koloni oluşturan yanıt testi ile taranması degrade Nanopattern imalatı

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.

Kızılötesi Lazer Destekli Mikropatterning ile Hücre Geometrisinin Kontrolü

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.

Mikrodesen Traksiyon Mikroskobu için Desen Oluşturma

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