D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

Tutte le procedure sono condotte a temperatura ambiente (~ 23 ° C), salvo diversamente indicato. 1. Fabbricazione di Silicon Maestro <ol><li> Pulire wafer di silicio con 100% di etanolo o xilene e secco sotto O 2 / N 2 gas. </li><li> Posizionare wafer di silicio in spin-coater a velocità di rotazione di 2.000-4.000 rpm per produrre un film di spessore 0,3-0,5 micron. </li><li> Reticolo la pellicola di fotoresist con le dimensioni corrette utilizzando un sistema di fotolitografia </li><li> Immergere completamente le fette di silicio photoresist rivestite con fantasia in un volume appropriato di soluzione photoresist in via di sviluppo. </li><li> Sciacquare i wafer di silicio photoresist rivestite sviluppati con acqua deionizzata. </li><li> Per formare array di sub-micron creste scala con pareti laterali verticali vicino, profondo reattiva etch ioni di silicio esposto utilizzando un sistema di incisione. </li><li> Rimuovere il fotoresist rimanente posizionando il wafer di silicio in un sistema asher plasma. </li><li> Tagliare il silicio wAfers con una fresa diamantata nei padroni di silicio opportunamente dimensionati per il successivo stampaggio replica. </li></ol> 2. Fabbricazione di PUA Stampo da Silicon Maestro NOTA: Volume da aggiungere al padrone silicio per nanofabbricazione varierà a seconda della zona del master nanostrutturati da replicare e la viscosità della soluzione di poliuretano acrilato (PUA). <ol><li> Pulire la superficie di silicio maestro con il 100% di etanolo o xilene e asciutto in O 2 / N 2 gas. </li><li> Posizionare lato silicio maestro modello in una capsula di Petri. </li><li> Per un maestro di silicio con 2 cm x 2 cm modello di superficie, pipetta 40 ml di PUA al modello di superficie. </li><li> Mettere un foglio di 4 cm x 4 cm trasparente in poliestere (PET) il PUA erogato. </li><li> Premere sul foglio PET e diffondere la PUA sotto il lenzuolo sulla faccia pattern usando un rullo o una superficie piana taglio (ad esempio una carta) in modo che l&#39;interamodello è coperto dal prepolimero PUA. </li><li> Mettere maestro di silicio, prepolimero, e PET circa 10 cm sotto una luce 20 Watt (115 V) UV (λ = 365 nm) per 50 sec. Per essere efficace, la lunghezza d&#39;onda della luce UV può essere ovunque tra 310-400 nm. L&#39;intensità della luce è 10-15 mW / cm 2 alla superficie del substrato. </li><li> Dopo l&#39;indurimento, rimuovere i fogli di PET lentamente con una pinza. PUA deve allegare al film PET con un negativo del silicio maestro nanopattern. </li><li> Cure nanopatterns PUA / PET in UV per almeno 12 ore prima dell&#39;uso. La sovraesposizione non è un problema. </li><li> Per pulire maestri silicio, posizionare un altro film di PET in cima al padrone senza l&#39;aggiunta di PUA e esporre a luce UV (λ = 365 nm) per 50 sec e rimuovere i fogli di PET. Questo eliminerà eventuali monomeri che non hanno reagito. </li><li> Sciacquare maestro di silicio con il 100% di etanolo o xilene e secco sotto O 2 / N 2 gas. </li></ol> 3a. Polymer nanopatterning poliuretano </p&gt; <ol><li> Preparare 25 mm di diametro vetrini circolari ponendo in un camera di trattamento ozono per 10 min. </li><li> Posizionare vetrini ozono trattati sul piccolo blocco PDMS per una facile movimentazione. </li><li> Applicare uno strato sottile di superficie promotore di adesione con pennello per vetrini. Vie vetrini asciugare per 30 min. </li><li> Posizionare il vetrino su un pezzo di carta per stampanti. </li><li> Goccia dispensare 10 ml di poliuretano (PU) pre-polimero (NOA 76) al centro di vetrino. Assicurarsi che non siano presenti bolle dopo l&#39;aggiunta. </li><li> Muffa posto PUA, modello a faccia in giù, sul vetrino. Disperdere il PU uniforme su tutta la superficie del vetrino tirando un rullo di gomma cilindro lungo lo stampo PUA. Carta per stampanti in grado di assorbire troppo pieno polimero. </li><li> Lampada UV Watt. Tempo di polimerizzazione del PU dipende dalla potenza della sorgente UV. </li><li> Togliere il campione dal sorgente di luce UV e staccare con cura lo stampo PUA dal vetrino spalmato PU. Polimerizzazione del PU è considerato complete quando i PUA stampo bucce pulito di distanza dal campione e il vetrino PU ha un aspetto cangiante. </li><li> Posizionare campioni finiti in essiccatore per la conservazione a lungo di un mese. </li></ol> 3b. Nanopatterning Poli (Lactide-Co-Glycolide) Hydrogel <ol><li> Creare uno stampo PDMS piatto vigorosamente mescolando silicone base di elastomero di silicone elastomero agente indurente in un rapporto di 10:1. </li><li> Pour PDMS misti precursore soluzione in una capsula di Petri in modo che il precursore PDMS raggiunge i 5 mm fino al bordo del piatto (cioè tale PDMS uno spessore di 5 mm). </li><li> Porre piastra di Petri e PDMS precursore in un essiccatore per 1 ora a Degas. </li><li> Spostare piastra di Petri e PDMS precursore in un forno a 65 ° C per almeno 2 ore per curare. </li><li> Dopo PDMS è guarito, utilizzare un rasoio per tagliare i PDMS piatto in 3 cm x 3 cm sezioni quadrate. Questi saranno i PDMS piatto stampi utilizzati in seguito nel processo di patterning. </li><li> Clean 25 mm di diametro vetrini circolare di placing in alcol isopropilico per 30 minuti in un sonicatore acqua. </li><li> Dry vetrini puliti in O 2 / N 2 gas. </li><li> Goccia dispensare 100 ul di soluzione di PLGA (15% w / v PLGA in cloroformio) sul vetrino. </li><li> Posizionare uno stampo PDMS piatta sulla parte superiore del PLGA erogato per assorbire il solvente e ottenere una superficie piana PLGA. Applicare una leggera pressione (~ 10 kPa) posizionando un peso di 200 g in cima alle PDMS per 5 min. </li><li> Sbucciare lentamente stampo PDMS piana e posizionare il vetro di copertura su un piatto preriscaldato (120 ° C) per 5 minuti per rimuovere il solvente residuo e aumentare l&#39;adesione tra il PLGA e il vetro di copertura. </li><li> Collocare lo stampo NP PUA sopra il piatto PLGA e applicare una pressione costante (~ 100 kPa) e calore (120 ° C) posizionando un peso 1 kg sulla parte superiore dello stampo PUA mentre sulla piastra di riscaldamento per 15 min. </li><li> Rimuovere il peso dal PLGA coperchio scorrevole e lasciare il substrato raffreddare a temperatura ambiente. Non rimuovere PUA stampo fino a quando i substrati sono statiraffreddato. </li><li> Una volta che i substrati sono sufficientemente raffreddato, togliere via lo stampo NP PUA, rivelando il substrato NP PLGA. Il substrato NP PLGA dovrebbe avere un aspetto iridescente. </li><li> Posizionare campioni finiti in essiccatore per la conservazione a lungo di un mese. </li></ol> 4. Semina cellulare e cultura NOTA: Questo protocollo descrive la cultura della neonatali miociti ventricolari di ratto (NRVMs) e cardiomiociti H7 umane staminali embrionali derivate da cellule (hESC-CMS), ma altre fonti di cellule possono essere utilizzati. <ol><li> Applicare i coprioggetti ANF (PU o PLGA) di un tessuto di cultura piatto di polistirolo da 35 mm. Pipettare 20 ml di Norland Optical Adhesive (NOA83H) sul fondo del piatto e posizionare con cura il vetrino ANF sulla parte superiore del NOA. Lasciare che la colla per stendere e coprire tutto il fondo vetrino. Cure NOA esponendo piatto ai raggi UV per 10 min. </li><li> Sterilizzare ANF risciacquando con 2 ml di soluzione acquosa di etanolo al 70% per 5 minuti, due volte. Rimuovere ethanol per aspirazione. Lasciare ANF completamente asciugare all&#39;aria per circa 1 ora sotto la lampada di sterilizzazione UV (λ = 200-290 nm) nella cappa di sicurezza biologica. </li><li> Adesione cellulare è arricchito da rivestimento della ANF in fibronectina durante la notte. Diluire fibronectina in acqua deionizzata a 5 mg / ml. Pipettare 2 ml di soluzione di fibronectina nel piatto. Porre in incubatore a 37 ° C e 5% CO 2 durante la notte (almeno 6 ore). </li><li> Ottenere NRVMs, hESC-CMS o altre cellule cardiache di interesse in base alle precedenti protocolli 22. </li><li> Campione di cellule centrifugare a 1000 rpm per 3 minuti per agglomerare le cellule. </li><li> Rimuovere con attenzione il surnatante tramite aspirazione. Assicurarsi di non disturbare il pellet. </li><li> Risospendere le cellule in opportuno terreno di coltura ad una concentrazione di 4,6 x 10 6 cellule / ml. </li><li> Pipetta con cautela 200 ml di sospensione cellulare su ANF sterilizzati. Assicurarsi sospensione cellulare rimane sul vetrino. </li><li> Posizionare cellule in incubatore a 37 ° C e 5%CO 2 per 4 ore per permettere alle cellule di allegare al ANF. </li><li> Aggiungere 2 ml di terreni di coltura caldo al piatto e sostituire le cellule in incubatrice alle stesse condizioni. </li><li> Dopo 24 ore, rimuovere il supporto e lavare con 2 ml di DPBS due volte per rimuovere le cellule in eccesso. </li><li> Aggiungere 2 ml di terreni di coltura caldo al piatto e sostituire le cellule in incubatrice alle stesse condizioni. Cultura le cellule a confluenza. Sostituire i media ogni altro giorno. </li></ol>

<ol>
	<li>Lozano, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+mortality+from+235+causes+of+death+for+20+age+groups+in+1990+and+2010:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2010.">Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010.</a> The Lancet. 380, 2095-2128 (2012).</li><li>Kim, D. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+cues+regulate+the+structure+and+function+of+macroscopic+cardiac+tissue+constructs.">Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs.</a> Proceedings of the National Academy of Sciences. 107, 565-570 (2010).</li><li>Tulloch, N. L., 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=Growth+of+Engineered+Human+Myocardium+With+Mechanical+Loading+and+Vascular+Coculture.">Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture.</a> Circulation Research. 109, 47-59 (2011).</li><li>Bursac, N., Parker, K., Irvanian, S., Tung, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+Cultures+With+Controlled+Macroscopic+Anisotropy:+A+Model+for+Functional+Electrophysiological+Studies+of+Cardiac+Muscle.">Cardiomyocyte Cultures With Controlled Macroscopic Anisotropy: A Model for Functional Electrophysiological Studies of Cardiac Muscle.</a> Circulation Research. 91, (2002).</li><li>Fink, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+stretch+of+engineered+heart+tissue+induces+hypertrophy+and+functional+improvement.">Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement.</a> The FASEB Journal. 14, 669-679 (2000).</li><li>Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+and+Engineering+the+Cell+Surface+Interface.">Exploring and Engineering the Cell Surface Interface.</a> Science. 310, (2005).</li><li>Mark, K., Park, J., Bauer, S., Schmuki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+engineering+of+biomimetic+surfaces:+cues+from+the+extracellular+matrix.">Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix.</a> Cell Tissue Res. 339, 131-153 (2009).</li><li>L&#252;, J. -. M., Wang, X., Marin-Muller, C., Wang, H., Lin, P. H., Yao, Q., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+research+and+clinical+applications+of+PLGA-based+nanotechnology.">Current advances in research and clinical applications of PLGA-based nanotechnology.</a> Expert. Rev. Mol. Diagn. 9, 325-341 (2009).</li><li>Kim, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Ann Biomed Eng. 40, 1339-1355 (2012).</li><li>Kim, D. -. H., Provenzano, P. P., Smith, C. L., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+nanotopography+as+a+regulator+of+cell+function.">Matrix nanotopography as a regulator of cell function.</a> The Journal of Cell Biology. 197, 351-360 (2012).</li><li>Park, J., Kim, H. -. N., Kim, D. -. H., Levchenko, A., Kahp-Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+the+Combined+Effect+of+Substrate+Rigidity+and+Topographic+Guidance+on+Cell+Morphology.">Quantitative Analysis of the Combined Effect of Substrate Rigidity and Topographic Guidance on Cell Morphology.</a> IEEE Trans.on Nanobioscience. 11, 28-36 (2012).</li><li>Kim, D. -. H., Lee, H., Lee, Y. K., Nam, J. -. M., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Nanopatterns+as+Enabling+Tools+for+Analysis+and+Control+of+Live+Cells.">Biomimetic Nanopatterns as Enabling Tools for Analysis and Control of Live Cells.</a> Adv. Mater. 22, 4551-4566 (2010).</li><li>Kim, D. -. H., Wong, P. K., Park, J., Levchenko, A., Sun, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineered+Platforms+for+Cell+Mechanobiology.">Microengineered Platforms for Cell Mechanobiology.</a> Annu. Rev. Biomed. Eng. 11, 203-233 (2009).</li><li>Kim, D. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopatterned+cardiac+cell+patches+promote+stem+cell+niche+formation+and+myocardial+regeneration.">Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration.</a> Integr Biol. 4, 1019 (2012).</li><li>Kim, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+nanostructures+of+polyethylene+glycol+for+applications+to+protein+adsorption+and+cell+adhesion.">Fabrication of nanostructures of polyethylene glycol for applications to protein adsorption and cell adhesion.</a> Nanotechnology. 16, 2420-2426 (2005).</li><li>Hwang, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+Assays+of+Endothelial+Cells+on+Nanopatterned+Surfaces+within+a+Microfluidic+Channel.">Adhesion Assays of Endothelial Cells on Nanopatterned Surfaces within a Microfluidic Channel.</a> Anal. Chem. 82, 3016-3022 (2010).</li><li>Heidenreich, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forecasting+the+Future+of+Cardiovascular+Disease+in+the+United+States:+A+Policy+Statement+From+the+American+Heart+Association.">Forecasting the Future of Cardiovascular Disease in the United States: A Policy Statement From the American Heart Association.</a> Circulation. 123, 933-944 (2011).</li><li>Legrice, I. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+structure+of+the+heart:+ventricular+myocyte+arrangement+and+connective+tissue+architecture+in+the+dog.">Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog.</a> Am J Physiol Heart Circ Physiol. 269, 1-12 (2002).</li><li>Sosnovik, D. E., Wang, R., Dai, G., Reese, T. G., Wedeen, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+MR+tractography+of+the+heart.">Diffusion MR tractography of the heart.</a> J Cardiovasc Magn Reson. 11, 47 (2009).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Fluxes+Involved+in+Control+of+Cardiac+Myocyte+Contraction.">Calcium Fluxes Involved in Control of Cardiac Myocyte Contraction.</a> Circulation Research. 87, 275-281 (2000).</li><li>Mohrman, D. E., Heller, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular Physiology. , (2010).</li><li>Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+De+Novo+Cardiomyocytes:+Human+Pluripotent+Stem+Cell+Differentiation+and+Direct+Reprogramming.">Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming.</a> Stem Cell. 10, 16-28 (2012).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Cardiomyocytes+Derived+From+Human+Induced+Pluripotent+Stem+Cells.">Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells.</a> Circulation Research. 104, (2009).</li><li>Qian, J. -. Y., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cytosolic+Ca2++dynamics+in+cultured+Guinea+pig+cardiomyocytes+as+an+in+vitro+model+to+identify+potential+cardiotoxicants.">Altered cytosolic Ca2+ dynamics in cultured Guinea pig cardiomyocytes as an in vitro model to identify potential cardiotoxicants.</a> Toxicology in Vitro. 24, 960-972 (2010).</li><li>You, M. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistically+Enhanced+Osteogenic+Differentiation+of+Human+Mesenchymal+Stem+Cells+by+Culture+on+Nanostructured+Surfaces+with+Induction+Media.">Synergistically Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells by Culture on Nanostructured Surfaces with Induction Media.</a> Biomacromolecules. 11, 1856-1862 (2010).</li><li>Kim, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopography-guided+tissue+engineering+and+regenerative+medicine.">Nanotopography-guided tissue engineering and regenerative medicine.</a> Advanced Drug Delivery Reviews. 65, 536-558 (2013).</li><li>Mannix, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomagnetic+actuation+of+receptor-mediated+signal+transduction.">Nanomagnetic actuation of receptor-mediated signal transduction.</a> Nature Nanotech. 3, 36-40 (2007).</li><li>Karuri, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+length+scale+topography+enhances+cell-substratum+adhesion+of+human+corneal+epithelial+cells.">Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells.</a> J Cell Sci. 117, 3153-3164 (2007).</li><li>Cavalcanti-Adam, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92, 2964-2974 (2007).</li><li>Koo, L. Y., Irvine, D. J., Mayes, A. M., Lauffenburger, D. A., Griffith, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-regulation+of+cell+adhesion+by+nanoscale+RGD+organization+and+mechanical+stimulus.">Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus.</a> J Cell Sci. 115, 1-11 (2002).</li><li>Yim, E. K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26, 5405-5413 (2008).</li><li>Dalby, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+human+mesenchymal+cell+differentiation+using+nanoscale+symmetry+and+disorder.">The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.</a> Nat Mater. 6, 997-1003 (2007).</li><li>Park, J., Bauer, S., Mark, v. o. n. . d. e. r. . K., Schmuki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosize+and+Vitality:+TiO+2Nanotube+Diameter+Directs+Cell+Fate.">Nanosize and Vitality: TiO 2Nanotube Diameter Directs Cell Fate.</a> Nano Lett. 7, 1686-1691 (2007).</li><li>Entcheva, E., Bien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+optical+mapping+of+excitation+in+cardiac+cell+networks+with+ultra-high+spatiotemporal+resolution.">Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.</a> Progress in Biophysics and Molecular Biology. 92, 232-257 (2006).</li><li>Tung, L., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+arrhythmias+in+tissue+culture.">Optical imaging of arrhythmias in tissue culture.</a> Journal of Electrocardiology. 39, (2006).</li><li>Himel, H. D., Bub, G., Lakireddy, P., El-Sherif, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+arrhythmias+in+the+cardiomyocyte+monolayer.">Optical imaging of arrhythmias in the cardiomyocyte monolayer.</a> Heart Rhythm. 9, 2077-2082 (2012).</li><li>Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+dynamic+in+vivo+environments+with+stimuli-responsive+materials+for+cell+culture.">Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture.</a> Trends in Biotechnology. 30, 426-439 (2012).</li><li>Henderson, D. J., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+and+Structure+of+the+Ventricles+in+the+Human+Heart.">The Development and Structure of the Ventricles in the Human Heart.</a> Pediatr Cardiol. 30, 588-596 (2009).</li><li>Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Micropatterned+Cardiac+Cell+Cultures+with+Realistic+Ventricular+Microstructure.">Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure.</a> Biophysj. 96, 3873-3885 (2009).</li><li>Badrossamay, M. R., McIlwee, H. A., Goss, J. A., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+Assembly+by+Rotary+Jet-Spinning.">Nanofiber Assembly by Rotary Jet-Spinning.</a> Nano Lett. 10, 2257-2261 (2010).</li><li>Rao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+microgrooved+culture+substrates+on+calcium+cycling+of+cardiac+myocytes+derived+from+human+induced+pluripotent+stem+cells.">The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells.</a> Biomaterials. 34, 2399-2411 (2013).</li></ol>

Authors have nothing to disclose.

Tessuti cardiaci funzionalmente maturi sono carenti sia in vivo che in vitro applicazioni di ingegneria tissutale cardiaco. I metodi di nanofabbricazione CFL qui descritte sono tecniche robuste per realizzare l&#39;allineamento cellulare e influenzare la funzione del tessuto macroscopico dovuto alla scalabilità del sistema. Grandi aree possono essere facilmente modellati e utilizzati per la coltura cellulare. Macroscopico allineamento cellulare è essenziale in ingegneria tessuto cardiaco per creare biomimetic...

Le malattie cardiovascolari sono la principale causa di morbilità e mortalità nel mondo e presentare un pesante fardello socio-economico su un già tesa globale 1,17 sistema sanitario. Ingegneria tissutale cardiaca ha due obiettivi distinti: (1) per rigenerare il miocardio danneggiato dopo la malattia ischemica o cardiomiopatia o (2) per creare un modello ad alta fedeltà del cuore per lo screening in vitro di droga o di modellazione malattia. Il cuore è un organo complesso che deve lavorare costantemente per la fornitura di sangue al corpo. Strutture laminari densamente di cardiomiociti e tessuti di sostegno sono disposti in schemi elicoidali tutta la parete cardiaca 18,19. Il cuore è anche accoppiato elettromeccanico 20 in un modo altamente coordinato per espellere efficacemente sangue al corpo 21. Diversi ostacoli principali restano da affrontare, tuttavia, prima intricato disegno della natura può attendibilmente essere ricapitolato in vitro.In primo luogo, anche se robusti metodi di differenziazione dei cardiomiociti continuano ad essere sviluppato 22, HPSC-CM esporre ancora fenotipi piuttosto immaturi. Le loro proprietà elettromeccaniche e la morfologia più strettamente corrispondono livelli fetali 23. In secondo luogo, se tenuto in condizioni di coltura tradizionali, sia derivato dalle cellule staminali e cardiomiociti primari non riescono ad assemblare in strutture native, tessuto-like. Piuttosto, le cellule diventano orientati in modo casuale e non presentano l&#39;aspetto a forma di bastoncino fasciato di miocardio adulto 24. La matrice (ECM) ambiente extracellulare con cui le cellule interagiscono gioca un ruolo importante in numerosi processi cellulari 11,13,25. L&#39;ECM consiste di complessi segnali molecolari e topografici, ben definiti che influenzano significativamente la struttura e la funzione delle cellule 6,26. All&#39;interno del cuore, l&#39;allineamento cellulare segue da vicino le fibre ECM sottostante scala nanometrica 2. L&#39;impatto di questi nanotopographspunti iCal sul cellulare e la funzione del tessuto, tuttavia, è ben lungi dall&#39;essere completamente capito. Studi preliminari di interazione cellula-biomateriale scala nanometrica indicano la potenziale importanza e l&#39;impatto di sub-micron spunti topografici per la segnalazione cellulare 27, 28-30 adesione, la crescita del 31, e la differenziazione 32,33. Tuttavia, a causa della difficoltà di sviluppare substrati nanofabricated riproducibili e scalabile, tali studi potrebbero non riprodurre gli effetti cellulari multi-scala del complesso in ambiente ECM vivo. In questo protocollo, una tecnica di nanofabbricazione semplice e conveniente per produrre scaffold di coltura cellulare che mimano allineamento delle fibre ECM cardiaca nativa è descritto, consentendo una vasta gamma di nuove ricerche di interazioni cardiomiociti-biomateriale. Capire come cardiomiociti interagiscono con l&#39;ambiente ECM nanoscala potrebbe consentire per la capacità di controllare il comportamento cellulare per imitare più da vicino funzioni tessuto nativozione. Inoltre, monostrati cellulari sono un sistema sperimentale semplificato rispetto a strutture 3D, ma ancora presentano un comportamento multicellulare complesso per le indagini penetranti e screening funzionale 2,34-36. Infine, tali impalcature potrebbero essere utilizzati per migliorare la funzione del trapianto cellulare quando impiantato nel cuore per fini rigenerativi 37.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<colgroup>
		<col span="4" style="width:66pt" width="88" />
	</colgroup>
	<tbody>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Name</td>
			<td class="xl63" style="width:66pt" width="88">Company</td>
			<td class="xl63" style="width:66pt" width="88">Catalogue number</td>
			<td class="xl64" style="width:66pt" width="88">Comments (optional)</td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fibronectin</td>
			<td class="xl65" style="width:66pt" width="88">BD Biosciences</td>
			<td class="xl65" style="width:66pt" width="88">354008</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 76</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">7606B</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl65" height="106" style="height:79.5pt;width:66pt" width="88">Surface Adhesion Promotor (Glass Primer)</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">PUA</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MINS-311RM</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Soft Rubber Roller</td>
			<td class="xl65" style="width:66pt" width="88">Speedball</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Silicon Wafers</td>
			<td class="xl65" style="width:66pt" width="88">NOVA Electronic Materials</td>
			<td class="xl65" style="width:66pt" width="88">FA01-9900</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Photoresist</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">SPRT510</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Photoresist Developer</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">MF320</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Electron-Beam Lithography System</td>
			<td class="xl65" style="width:66pt" width="88">JEOL</td>
			<td class="xl65" style="width:66pt" width="88">JBX-9300FS</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Etching System</td>
			<td class="xl65" style="width:66pt" width="88">Surface Technology Systems</td>
			<td class="xl65" style="width:66pt" width="88">NP10 8UJ</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Plasma Asher System</td>
			<td class="xl65" style="width:66pt" width="88">BMR Technology Co.</td>
			<td class="xl65" style="width:66pt" width="88">DSF-200</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Ozone Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-O- 08</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fusion Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-A 11</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 83H</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">8301</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Spin Coater</td>
			<td class="xl65" style="width:66pt" width="88">Laurel Technology</td>
			<td class="xl65" style="width:66pt" width="88">WS-400-6NPP</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Skyrol PET Film</td>
			<td class="xl65" style="width:66pt" width="88">SKC Co., Ltd.</td>
			<td class="xl65" style="width:66pt" width="88">23038-59-9</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">25mm Glass Slides</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">2948</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Sylgard 184 Silicone Elastomer Kit</td>
			<td class="xl65" style="width:66pt" width="88">Dow Corning</td>
			<td class="xl67" style="width:66pt" width="88">6/5/2553</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Poly(D,L-lactide-co-glycolide)</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">P2191-1G</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Chloroform</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">372978-1L</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
			<td class="xl65" style="width:66pt" width="88">Global Insustrial</td>
			<td class="xl65" style="width:66pt" width="88">T9FB503120</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Isopropyl Alcohol</td>
			<td class="xl65" style="width:66pt" width="88">EMD Millipore</td>
			<td class="xl65" style="width:66pt" width="88">PX1835-2</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Hot Plate</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">PC-420D</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Sonicator</td>
			<td class="xl65" style="width:66pt" width="88">Branson</td>
			<td class="xl65" style="width:66pt" width="88">B2510MTH</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
	</tbody>
</table>

capillary force lithography for cardiac tissue engineering

La malattia cardiovascolare resta la principale causa di morte nel mondo 1. Ingegneria tissutale cardiaca molto promettente per fornire scoperte mediche rivoluzionarie con gli obiettivi di sviluppo di tessuti funzionali per la rigenerazione cardiaca, così come test di screening in vitro. Tuttavia, la capacità di creare modelli ad alta fedeltà di tessuto cardiaco è rivelato difficile. Matrice extracellulare del cuore (ECM) è una struttura complessa costituita da entrambi i segnali biochimici e biomeccanici che vanno dalla micro-alla scala nanometrica 2. Locali condizioni di carico meccanico e le interazioni cellula-ECM sono stati recentemente riconosciuti come componenti vitali in ingegneria del tessuto cardiaco 3-5. Una gran parte della ECM cardiaco è composto da fibre di collagene allineate con diametri scala nanometrica che influenza significativamente l&#39;architettura dei tessuti e accoppiamento elettromeccanico 2. Purtroppo, alcuni metodi have stato in grado di mimare l&#39;organizzazione delle fibre ECM fino alla scala nanometrica. Recenti progressi nelle tecniche di nanofabbricazione, tuttavia, hanno permesso la progettazione e la realizzazione di ponteggi scalabili che imitano i segnali vivo rigidità strutturali e substrato della ECM nel cuore 6-9. Presentiamo qui lo sviluppo di due riproducibile, processi nanopatterning scalabili per l&#39;allineamento funzionale delle cellule cardiache utilizzando la poli polimero biocompatibile (lattide-co-glicolico) (PLGA) 8 e un poliuretano (PU) polimero a base conveniente, e. Questi substrati anisotropicamente nanofabricated (ANF) imitare la ECM sottostante ben organizzate, tessuti allineati e può essere utilizzato per studiare il ruolo di nanotopography sulla morfologia e la funzione delle cellule 10-14. Utilizzando un nanostrutturati (NP) maestro di silicio come un modello, un acrilato di poliuretano (PUA) muffa è fabbricato. Questo stampo PUA viene quindi utilizzato per pattern l&#39;idrogel PU o PLGA tramite solventi mediata litografia forza capillare (CFL)-UV assistita o, rispettivamente, 15,16. In breve, PU o PLGA pre-polimero è caduta erogato su un vetrino di vetro e lo stampo PUA è posto sulla parte superiore. Per CFL-UV assistita, il PU viene poi esposto a radiazioni UV (λ = 250-400 nm) per la polimerizzazione. Per mediata solvente CFL, il PLGA viene stampato mediante calore (120 ° C) e pressione (100 kPa). Dopo la polimerizzazione, lo stampo PUA è staccata, lasciando dietro un ANF per coltura cellulare. Pile, come neonatali miociti ventricolari di ratto, così come cardiomiociti derivati ​​dalle cellule staminali umane pluripotenti, possono essere mantenuti in ANF 2.

La figura 1 è uno schema del processo di produzione per i due metodi di fabbricazione. A causa della diffrazione della luce causata dalla topografia nanoscala, nanopatterning dovrebbe risultare in una superficie iridescente alla ANF. Figura 2 mostra questa superficie iridescente su un ben modellata-25 millimetri coprioggetto NP-PU (Figura 2A) con cresta 800 nm e scanalatura larghezza (Figura 2B). L&#39;aspetto iridescente della ANF varierà leggermente...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

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 ...

capillary-force-lithography-for-cardiac-tissue-engineering

Research

JoVE Journal

Bioengineering

12.3K Views.  University of Washington. 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.

Video: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Fabbricazione di derivazione biologica Materiali iniettabili per l&#39;ingegneria tissutale miocardica

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Ricerca

Bioingegneria

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plasma Surface Patterning litografia per la creazione di reti di cella

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Incapsulamento di cardiomiociti in un idrogel di fibrina per l&#39;ingegneria tissutale cardiaca

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Elaborazione Postproduzione di fibre elettrofilate per l&#39;ingegneria tissutale

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparazione di Thermoresponsive Nanostrutturati Superfici Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Un protocollo per Decellularizing Mouse coclee per l'ingegneria dei tessuti dell'orecchio interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...