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 porzi...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>CO2 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&#8217;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&#160;</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-scrit...

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

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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Ricerca

Bioingegneria

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 ...

A Microfluidic Technique to Probe Cell Deformability

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

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

Una tecnica Microfluidic per sondare deformabilità cellulare

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

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Realizzazione substrati di coltura complessi utilizzando robotica microcontact stampa (R-μCP) e sequenziale Sostituzione nucleofila

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una tecnica senza etichetta per l&#39;Imaging spazio-temporale delle secrezioni monocellulari

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Selective cellulare eliminazione dalla misto Cultura 3D utilizzando una tecnica Near Infrared Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Misura di rigidità dei substrati in morbido Silicone per gli studi di Mechanobiology usando un microscopio a fluorescenza Widefield

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Modellazione della geometria delle colonie di cellule staminali embrionali umane su substrati conformi per controllare la meccanica a livello di tessuto

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Formulazione e modulazione acustica di nanogoccioline di perfluorocarburi vaporizzate otticamente

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

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

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Patterning cellulare su fotolitograficamente Definito parylene-C: SiO

Una tecnica Microfluidic per sondare deformabilità cellulare

Realizzazione substrati di coltura complessi utilizzando robotica microcontact stampa (R-μCP) e sequenziale Sostituzione nucleofila

Una tecnica senza etichetta per l'Imaging spazio-temporale delle secrezioni monocellulari

Selective cellulare eliminazione dalla misto Cultura 3D utilizzando una tecnica Near Infrared Photoimmunotherapy

Misura di rigidità dei substrati in morbido Silicone per gli studi di Mechanobiology usando un microscopio a fluorescenza Widefield

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

Modellazione della geometria delle colonie di cellule staminali embrionali umane su substrati conformi per controllare la meccanica a livello di tessuto

Formulazione e modulazione acustica di nanogoccioline di perfluorocarburi vaporizzate otticamente

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO

Una tecnica Microfluidic per sondare deformabilità cellulare

Realizzazione substrati di coltura complessi utilizzando robotica microcontact stampa (R-μCP) e sequenziale Sostituzione nucleofila

Una tecnica senza etichetta per l'Imaging spazio-temporale delle secrezioni monocellulari

Selective cellulare eliminazione dalla misto Cultura 3D utilizzando una tecnica Near Infrared Photoimmunotherapy

Misura di rigidità dei substrati in morbido Silicone per gli studi di Mechanobiology usando un microscopio a fluorescenza Widefield

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

Modellazione della geometria delle colonie di cellule staminali embrionali umane su substrati conformi per controllare la meccanica a livello di tessuto

Formulazione e modulazione acustica di nanogoccioline di perfluorocarburi vaporizzate otticamente

La creazione di substrati Patterned bidimensionali per confinamento di proteine e cellule