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.

1. La replica del PDMS Stampo di uno stampo EBL <ol><li> Realizzare stampo in silicone 29 <ol><li> cappotto Spin 200 microlitri polimetilmetacrilato (PMMA) soluzione su un substrato 1 cm x 1 cm di silicio (Si) a 2.500 rpm per 1 min per formare una pellicola sottile. </li><li> Cuocere il film PMMA sul substrato di Si a 180 ° C per 2 min. </li><li> Scrivere il nanopattern disegnato sulla pellicola PMMA utilizzando un fascio elettronico focalizzato ad una dose di 300 uC / cm 2 zona. </li><li> Sviluppare la nanopattern PMMA in sviluppo per 80 sec. </li><li> Depositare lo nanopattern PMMA con uno strato di nichel di 50 nm di spessore usando un evaporatore E-fascio in uscita una tensione di 10 kV, corrente di emissione di 0,5 mA e una velocità di deposizione di 0,5 Å / sec. </li><li> Sollevare la parte PMMA in 20 ml di rimozione a 80 ° C per 20 min. </li><li> Reactive etch ioni (RIE) il nanopattern nel substrato di Si per ottenere uno stampo Si di profondità desiderata. NOTA: la miscela di gas di tetrafluoromethane (CF 4) / ossigeno (O 2) (90% / 10%) ad un plasma accoppiato induttivamente (ICP) potenza di 400 W e RIE potenza di 150 W viene utilizzato per incidere substrato di Si ad una profondità di 560 nm. </li></ol></li><li> muffa silanizzata Si <ol><li> Mettere un vetrino di vetro e lo stampo di Si in da 100 mm piatto di PS di Petri e trasferirle in un essiccatore di vetro situato in una cappa aspirante. </li><li> Goccia 10 ml 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane sul vetrino. Attenzione: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane può causare corrosione della pelle e gravi lesioni oculari. Indossare dispositivi di protezione adeguati (DPI). </li><li> Coprire la piastra di Petri parzialmente. </li><li> Mantenere un essiccatore sotto vuoto per 5 ore in una cappa aspirante per completare la silanizzazione dello stampo Si. </li></ol></li><li> Preparare PDMS prepolimero <ol><li> Pesare 10 g di resina PDMS e 1,05 g vulcanizzante in una barca pesata monouso. </li><li> Mescolare il prepolimero PDMS accuratamente utilizzando un cucchiaio di plastica. </li><li> Degassare la prepolimero PDMS in un essiccatore di plastica sotto vuoto per circa 20 minuti fino a quando si osserva una miscela chiaro. </li></ol></li><li> Replicare stampi PDMS <ol><li> Mettere lo stampo Si silanizzata in 60 millimetri piastra di Petri. </li><li> Versare il prepolimero PDMS sullo stampo Si nella piastra di Petri. </li><li> Posizionare la piastra di Petri in un essiccatore di plastica e Degas per circa 10 minuti fino a quando tutte le bolle scompaiono. </li><li> Trasferire la piastra di Petri a una piastra e curare il prepolimero PDMS a 70 ° C per 4 ore. </li><li> Staccare lo stampo PDMS dallo stampo Si accuratamente utilizzando una pinzetta. NOTA: stampi PDMS possono essere conservati in condizioni ambiente per un massimo di una settimana. Dopo il trattamento, ci sono alcune molecole di resina PDMS non reticolato e agente indurente residui negli stampi PDMS 30. Le molecole a basso peso molecolare gradualmente diffondere fuori e si accumulano in superficie nel tempo. Questo influisce sulle proprietà topografiche e meccaniche della superficie PDMS 31. DIFla fusione non è significativo entro una settimana. </li></ol></li></ol> 2. cucitura di PDMS Stampi in una grande, Mold singolo <ol><li> Preparare multipli PDMS stampi ripetendo passo 1.4. NOTA: Pesare stessa quantità di miscela PDMS ogni momento di ottenere stampi PDMS di uguale spessore. </li><li> Determinare l&#39;orientamento anisotropi PDMS nanopatterns come nanogratings sotto un microscopio ottico e segnare sul retro degli stampi PDMS con un pennarello. NOTA: Non è necessario marcare l&#39;orientamento nanotopography isotropo come nanopillars. </li><li> Pulire un substrato di Si con etanolo in una cappa aspirante e asciugarlo con aria compressa. </li><li> Tagliare le aree nanostrutturata di ogni stampo PDMS con una lama. NOTA: Per gli stampi PDMS che verranno messi alla periferia dello stampo cucito, solo le aree nanostrutturata a contatto con altri dovrebbero essere tagliati fuori. </li><li> Mettere lo stampo PDMS rifinito con la nanopattern a faccia in giù sulato specchio del substrato di Si e quindi allineare altri stampi vicino a, ma non toccare lo stampo circostante (s). </li><li> Preparare uno strato adesivo PDMS <ol><li> Cast 1 g degassata PDMS prepolimero (resina PDMS e curare rapporto agente: 10: 1,05) su un vetrino pulito (7,5 cm × 2,5 cm) per formare uno strato dello spessore di 0,5 mm. </li><li> Cuocere lo strato PDMS a 100 ° C su una piastra per 3-5 min. Utilizzare un ago per toccare il livello e garantire che il livello è parzialmente ma non completamente guarita. NOTA: PDMS parzialmente indurito non può fluire come affinati prepolimero PDMS, ma è appiccicoso confronto con PDMS curato. </li></ol></li><li> Posizionare lo strato PDMS sul retro di PDMS stampi allineati e invertire rapidamente questa assemblea e trasferirlo alla piastra. </li><li> Applicare una forza di compressione (5 kPa) utilizzando un blocco di metallo sulla parte superiore del complessivo per assicurare un buon contatto tra lo strato adesivo PDMS e retro di stampi PDMS, e curare le PDMS strato adesivo a 100 ° C per 1 ora. &lt;br /&gt; NOTA: regolare accuratamente la posizione del blocco metallico per evitare l&#39;inclinazione del complesso. </li><li> Rimuovere il blocco di metallo e staccare il singolo, di stampo PDMS cucita dal substrato di Si. </li></ol> 3. Generazione di uno stampo master su PS substrati Nota: Lo stampo PDMS cucita immobilizzato su un vetrino può essere utilizzato per generare uno stampo master su un piatto di PS o un film sottile PS, da cui possono essere prodotti lavorativi substrati nanostrutturati. <ol><li> Generare uno stampo master su un piatto di PS <ol><li> Preparare un piatto di PS <ol><li> Essiccare i granuli PS in un forno sotto vuoto a 80 ° C per due giorni. </li><li> Preriscaldare una pressa a 230 ° C. </li><li> Assemblare una piastra di alluminio, un foglio di politetrafluoroetilene (PTFE) e il distanziatore di alluminio in un ordine dal basso verso l&#39;alto. </li><li> Carico 3,5 g pellets PS del distanziatore di alluminio con una apertura quadrata di 3 cm (L) × 3 cm (W) × 0,3 cm (H). NOTA: Il distanziatore è di circa 0.1 cm di spessore maggiore di stampi PDMS, e quindi il substrato finale nanostrutturati PS ha uno spessore di circa 0,1 cm. </li><li> Posizionare un altro foglio di PTFE e poi un altro piatto di alluminio sul distanziatore di alluminio. </li><li> Posizionare il gruppo nella macchina stampa. </li><li> Preriscaldare i pellet PS a 230 ° C per 30 min. </li><li> Applicare una pressione di compressione (0,1 MPa) sul gruppo per 5 min. </li><li> Rilasciare la pressione e poi riapplicare una pressione di compressione di 0,5 MPa sul gruppo. </li><li> Ripetere il passaggio 3.1.1.9 con un aumento di pressione di 0,5 MPa fino alla pressione desiderabile di 1,5 MPa è raggiunto. </li><li> Spegnere il riscaldatore della macchina pressa e raffreddarlo sotto i 70 ° C ad una pressione costante di 1,5 MPa. </li><li> Prendere l&#39;assieme e conservare la piastra di PS in stufa da vuoto a 80 ° C per evitare che l&#39;umidità rientrare la piastra PS. </li></ol></li><li> Nanoimprint lo stampo PDMS cucito in piatto di PS <ol><li> Posizionare la piastra di PS in un distanziale in alluminioimpostato su 3 pollici Si wafer. NOTA: Le dimensioni interne del distanziale sono uguali a quelli della lastra PS modo la piastra PS adatta a destra nel distanziatore. </li><li> Riscaldare la lastra PS su una piastra riscaldante a 250 ° C per 30 min. </li><li> Mettere lo stampo PDMS cucita con nanopatterns a faccia in giù sulla lastra PS fuso. NOTA: Un lato dello stampo PDMS è messo in contatto con la superficie della lastra PS primo e un altro lato si abbassa progressivamente a contatto con la superficie PS per evitare la formazione di bolle d&#39;aria all&#39;interfaccia. Attenzione: La superficie della piastra è caldo. Indossare thermogloves durante il processo nanoimprinting. </li><li> Inserire una piastra di alluminio sul vetrino dello stampo PDMS cucita. </li><li> Applicare una pressione di compressione (12,5 kPa) utilizzando blocchi di metallo sulla piastra di alluminio e attendere 3 min. NOTA: Assicurarsi che la piastra di alluminio non sia inclinato. </li><li> Sollevare e sostituire il blocco di metallo dalla piastra di alluminio e ioaumentare l pressione di compressione a 25 kPa. </li><li> Ripetere il passaggio 3.1.2.6 con la pressione aumentata a 50 kPa. NOTA: Questo passaggio è quello di rimuovere l&#39;aria intrappolata tra lo stampo PDMS e la piastra di PS. </li><li> Mantenere la temperatura della piastra tra 240 e 250 ° C sotto la pressione costante di 50 kPa per 15 min. </li><li> Spegnere la piastra e raffreddare l&#39;intero setup. NOTA: La ventola può essere utilizzato per accelerare il processo di raffreddamento. </li><li> Rimuovere i blocchi di metallo dopo che la temperatura è inferiore a 50 ° C, e con attenzione staccare lo stampo PDMS cucita dalla piastra di PS. NOTA: Il substrato PS ha le nanopatterns inversa e può essere usato come stampo master per produrre lavoro PDMS substrati. </li></ol></li></ol></li><li> Generare uno stampo master su un film sottile PS <ol><li> Preparare una sottile pellicola PS <ol><li> Sciogliere 1 g PS in 10 ml di toluene in una cappa aspirante. Attenzione: Toluene può causare irritatio pellen e gravi lesioni oculari, e possono provocare danni agli organi in caso di esposizione prolungata o ripetuta. Indossare adeguato DPI. </li><li> Spin-coat soluzione PS 1 ml su un wafer 2-in a 2.500 rpm per 1 min per formare ~ 1 micron di spessore PS film sottile. </li><li> Evaporare toluene dal film impostando il film PS su Si wafer in una cappa aspirante per 3 giorni. </li><li> Ricuocere il film sottile PS in stufa da vuoto a 80 ° C per una notte. </li></ol></li><li> Nanoimprint lo stampo PDMS su una sottile pellicola PS <ol><li> Mettere lo stampo PDMS cucita con nanotopography a faccia in giù sul film sottile PS, che è impostato su una piastra. </li><li> Applicare una pressione di compressione di 12 kPa sullo stampo PDMS utilizzando blocchi di metallo sul lato di vetro dello stampo PDMS. </li><li> Aumentare la temperatura della piastra riscaldante a 180 ° C e mantenere per 15 min. Attenzione: La pellicola PS fuso può funzionare come lubrificante. Prestare attenzione per evitare che i blocchi di metallo di scivolare fuori. </li><li> Spegnere la piastrae raffreddare l&#39;intero setup. NOTA: La ventola può essere utilizzato per accelerare il processo di raffreddamento. </li><li> Rimuovere i blocchi di metallo dopo che la temperatura scende al di sotto di 50 ° C, e attentamente staccare lo stampo PDMS cucita dal film PS. NOTA: Il film nanostrutturati PS servirà come uno stampo maestro per produrre substrati PDMS di lavoro. </li></ol></li></ol></li></ol> 4. Nanotopographical Modulazione di Cell Comportamento Nota: le cellule epiteliali umane sono coltivate sulle nanotopographies rappresentativi per dimostrare la modulazione nanotopographical di diffusione delle cellule. <ol><li> Cast substrati lavoro PDMS dallo stampo matrice generata da entrambi Fase 3.1 o 3.2 a seconda dell&#39;applicazione. </li><li> Utilizzando una conca arco in acciaio pugno, tagliare i PDMS nanostrutturati substrati in dischi per adattare la configurazione di un piatto multi-ben specifici (ad esempio targa, 24 pozzetti). </li><li> Utilizzare le pinzette per posizionare i dischi PDMS nei pozzetti oFa piatto multi-bene. </li><li> Sterilizzare i substrati PDMS utilizzando etanolo al 70% e quindi l&#39;esposizione UV, ciascuno per 30 min. </li><li> Lavare le PDMS substrati con 1x fosfato sterile salina tamponata (PBS) per tre volte. </li><li> Rivestire le PDMS substrati con proteine della matrice extracellulare (cioè, 20 ug / ml fibronectina) per 30 min a temperatura ambiente. </li><li> Sciacquare i PDMS substrati tre volte con PBS sterile, ciascuno per 5 min. </li><li> Sospendere A549 delle cellule del cancro del polmone umano in media aquila Dulbecco modificato con il 10% di siero fetale bovino e contare le cellule utilizzando un emocitometro. </li><li> Piastra le cellule ad una densità di semina di 2.000 cellule / cm 2 sui PDMS substrati e li coltura a 37 ° C in atmosfera umidificata contenente il 5% di CO 2 per un giorno. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Fissare le cellule in una miscela di 4% paraformaldeide e glutaraldeide al 2% in PBS per 4 ore e disidratare le cellule utilizzando un CO 2 PO criticaint asciugatrice per la scansione elettronica osservazione al microscopio 29. Attenzione: Paraformaldeide e glutaraldeide possono causare gravi ustioni cutanee e gravi lesioni oculari. Operare in una cappa chimica e all&#39;usura adeguata DPI. </li></ol>

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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.

Vi presentiamo un metodo semplice, economico, ma versatile per generare una vasta area di substrato nanostrutturati. Per espandere fedelmente nanopatterns altamente definite, grande attenzione deve essere rivolta a diversi passaggi critici. Il primo è quello di tagliare le molteplici stampi PDMS. aree nanostrutturata degli stampi PDMS devono essere rimossi. Inoltre, le pareti laterali degli stampi devono essere tagliati verticalmente perfetto possibile per minimizzare gli spazi tra gli stampi. Collettivamente, la porzione di aree nanostrutturata nello stampo del punto finale può essere ridotto. In secondo luogo, la superficie nanostrutturati di questi PDMS stampi deve essere allineata senza distorsioni sul substrato di silicio. Poiché le PDMS nanostrutture sono inclini a deformarsi, è fondamentale per posizionare le superfici nanostrutturati contro un lato specchio del substrato di silicio delicatamente e uniformemente (evitare di intrappolare aria tra lo stampo PDMS e superficie di silicio). Gli stampi PDMS saranno allineate il più vicino possibile, ma non tocca il niaghboring stampi per minimizzare ulteriormente la porzione nanostrutturata dello stampo punto finale. In caso contrario, le nanostrutture toccati si deformano durante nanoimprinting. In terzo luogo, lo spessore degli stampi PDMS può variare da lotto a lotto, e quindi è fondamentale per uniformare lo spessore oltre a rendere lo spessore di ogni uniforme stampo livellando il piccolo stampo Si perfettamente prima PDMS colata. Anche se la variazione di spessore di tutti gli stampi PDMS può essere compensato regolando lo spessore del prepolimero (adesivo) PDMS strato gettato su un vetrino, uno strato prepolimero spessore potrebbe essere problematico. Il prepolimero può essere tirato attraverso gli interstizi tra gli stampi PDMS alla superficie modellata dalla forza capillare, e di conseguenza danneggiare i nanopatterns. La variazione di spessore può essere minimizzato preparare la stessa quantità di miscela PDMS quando lancia dallo stampo EBL. Come risultato, un sottile strato di prepolimero PDMS può essere utilizzato. In alternativa, parzialmente curare la prepolymer strato aumenterà la viscosità, e quindi ridurre la sua raise ed eventualmente eliminare il possibile danneggiamento delle superfici nanostrutturati. La tecnica del punto è limitata dalla natura elastomerica del PDMS. Sebbene litografia soft è stato applicato per replicare dimensioni caratteristiche piccolo come 2 nm 32 e, in linea di principio, può raggiungere una risoluzione di meno di 0,5 nm 18, le caratteristiche PDMS nanoscala non possono essere replicate perfettamente quando il rapporto tra altezza e larghezza è troppo alta (&gt; 2) o troppo bassa (&lt;0,2). I nanofeatures può crollare quando il formato è troppo alto, o provocano sollievo insufficiente quando il timbro PDMS di un &lt;rapporto di 0,2 viene utilizzato 33. Inoltre, più PDMS stampi non possono essere cuciti senza causa degli interstizi e rifilatura incompleta degli stampi PDMS, e quindi non vi sono aree nanostrutturata e disallineati (in particolare per nanopatterns continui come nanogratings). Data la piccola percentuale della Defectedzona sopra la superficie totale, la tecnica del punto ancora fornisce un modo semplice e conveniente per produrre una grande area di substrati nanostrutturati. Inoltre, quando lo stampo viene cucita nanoimprinted nel substrato di polimero, polimero fuso possa scorrere nell&#39;interstizio, causando una superficie irregolare (Figura 1a). La superficie irregolare rende difficile per raccogliere campioni per analisi biologiche cellulari o molecolari. Nelle applicazioni di microfluidica, l&#39;aumento provoca anche una sigillatura incompleta quando microcanali sono sigillati contro il substrato fantasia. Il problema superficie irregolare può essere facilmente risolto applicando polimero tecnica del film sottile per minimizzare l&#39;aumento attraverso sintonizzazione lo spessore del film (Figura 1b). Anche se la tecnica del punto ha bisogno di uno stampo master definito per l&#39;espansione, è semplice e conveniente rispetto ad altre tecniche quali la litografia step-and-flash e un nanoimprinting lithogra roll-to-rollPHY. La tecnica di cucitura richiede solo piastre elettriche e un modo di esercitare forze di compressione durante i processi di cucitura e nanoimprinting, ma non costose attrezzature. Inoltre, il processo di cucitura può essere condotta in un ambiente pulito, ma non necessariamente in una camera sterile. La tecnica punto è anche versatile. Oltre ad ampliare un nanopattern identica ad una vasta area, la tecnica del punto può essere applicato per gli stampi costituiti caratteristiche micro e / o nanoscala di varie forme, dimensioni e disposizioni. A questo proposito, una biblioteca combinatoria di micro / nanotopographies può essere costruito per fornire la piattaforma high-throughput per indagare le interazioni cellula-topografia. Questa tecnica di punto semplice, conveniente e versatile può potenzialmente essere esteso per creare dispositivi micro / nanoscala con componenti ibridi.

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> La tecnica del punto può generare una grande superficie di substrati nanostrutturati con alta fedeltà. Figura 1a e 1b visualizzare la grande area di nanopatterns trasferite dallo stampo PDMS cucita piastra PS e PS film sottile su un substrato di Si, rispettivamente. Il confronto tra lo stampo originale EBL-scritta (figura 1c) e le PDMS finali lavorativi substrato (Figura 1d) conferma che le nanopatterns EBL-scritte possono essere fedelmente trasferiti al substrato lavorare. Nanotopography di varie geometrie e dimensioni può essere utilizzato per modulare il comportamento cellulare. Come mostrato nella Figura 2 con A549, una linea di cellule epiteliali basali adenocarcinomic come cellule modello, le nanogratings anisotropi può allungare le cellule lungo la direzione nanograting confronto con la morfologia multipolare che le cellule A549 visualizzare sul nanopillars isotropo. nt &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/54840/54840fig1.jpg" /> Figura 1. Generazione di una vasta area di substrati nanostrutturati usando la tecnica del punto. (A, b) le immagini ottiche delle nanopatterns trasferiti al piatto di PS e PS film sottile, rispettivamente. Le frecce indicano l&#39;aumento del polimero negli interstizi degli stampi PDMS cucite. (C, d) immagini SEM delle nanopatterns su stampo EBL e PDMS finali substrato di lavoro, rispettivamente. Le barre di scala sono 1 micron. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54840/54840fig2.jpg" /> Figura 2. Nanotopographical modulata cella diffusione delle cellule A549. </strong&gt; Cellule A549 coltivate su (a) nanogratings di 500 Nm a spessore della linea, 500 nm a distanza e 560 nm in altezza e (b) nanopillars di 500 nm di diametro, 450 nm a distanza da bordo a bordo e 560 nm in altezza rispettivamente. Le barre di scala sono 10 micron. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</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.

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

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.

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

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

Ricerca

Biologia dello sviluppo

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.

Capillare Forza litografia per Cardiac Tissue Engineering

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

Bioingegneria

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.

Microfabrication di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

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.

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO<sub&gt; 2</sub&gt; Substrati

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.

Stretching celle micropatterned su una membrana 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.

Micropatterning a cella singola semplice senza litografia con stencil laser-cut

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.

Un metodo versatile di patterning proteine ​​e cellule

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.

Fabbricazione di GNSM gradiente termico Nanoimprinting tecnica e Screening della risposta delle cellule endoteliali umane formanti colonie

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.

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

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.

Generazione di modelli per microscopia a trazione micropattern

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

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

Riepilogo

Esplora Altri Video

Capitoli in questo video

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Capillare Forza litografia per Cardiac Tissue Engineering

Microfabrication di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO

Stretching celle micropatterned su una membrana PDMS

Micropatterning a cella singola semplice senza litografia con stencil laser-cut

Un metodo versatile di patterning proteine e cellule

Fabbricazione di GNSM gradiente termico Nanoimprinting tecnica e Screening della risposta delle cellule endoteliali umane formanti colonie

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

Generazione di modelli per microscopia a trazione micropattern

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

Riepilogo

Esplora Altri Video

Capitoli in questo video

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Capillare Forza litografia per Cardiac Tissue Engineering

Microfabrication di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO

Stretching celle micropatterned su una membrana PDMS

Micropatterning a cella singola semplice senza litografia con stencil laser-cut

Un metodo versatile di patterning proteine ​​e cellule

Fabbricazione di GNSM gradiente termico Nanoimprinting tecnica e Screening della risposta delle cellule endoteliali umane formanti colonie

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

Generazione di modelli per microscopia a trazione micropattern

Un metodo versatile di patterning proteine e cellule