Name: fabrication of gradient nanopattern by thermal nanoimprinting technique and screening of the response of human endothelial colony-forming cells
Uploaded: 2018-07-01T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells at JoVE.com

Questo lavoro è stato supportato dal programma di ricerca di scienza base attraverso la nazionale Ricerca Fondazione della Corea (NRF) finanziato dal Ministero dell'istruzione, scienza e tecnologia (MEST) [NRF-2015R1D1A1A01060397] e Bio & Medical Technology Development Programma della NRF finanziato dal Ministero della scienza, ICT & futuro pianificazione [NRF-2017M3A9C6029563].

Questo studio è stato approvato dalla Institutional Review Board presso ospedale di Corea Università Anam (IRB No. ED170495). Tutte le procedure sono state effettuate conformemente alla dichiarazione di Helsinki e sue modifiche successive.
1. preparazione del substrato di alluminio (Al) di elettrolucidatura
Attenzione: Soluzione di elettrolucidatura è corrosivo e tossico. Indossare indumenti protettivi, compresi guanti di nitrile, occhiali e camice...

<ol>
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M., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+state+of+the+extracellular+matrix+regulates+the+structure+and+molecular+composition+of+cell-matrix+adhesions.">Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions.</a> Molecular biology of the cell. 11 (3), 1047-1060 (2000).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+growth+and+inflammatory+response+of+macrophages+and+foam+cells+with+nanotopography.">Control of growth and inflammatory response of macrophages and foam cells with nanotopography.</a> Nanoscale research letters. 7 (1), 394 (2012).</li><li>Kyle, D. J., Oikonomou, A., Hill, E., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+functional+evaluation+of+biomimetic+silicone+surfaces+with+hierarchical+micro/nano-topographical+features+demonstrates+favourable+in+vitro+foreign+body+response+of+breast-derived+fibroblasts.">Development and functional evaluation of biomimetic silicone surfaces with hierarchical micro/nano-topographical features demonstrates favourable in vitro foreign body response of breast-derived fibroblasts.</a> Biomaterials. 52, 88-102 (2015).</li><li>Seo, H. R., Joo, H. J., Kim, D. H., Cui, L. H., Choi, S. C., Kim, J. H., Cho, S. W., Lee, K. B., Lim, D. 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J., Jung, T., Kim, J. S., Lee, K. B., Chung, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographical+control+for+maintaining+undifferentiated+human+embryonic+stem+cell+colonies+in+feeder+free+conditions.">Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions.</a> Biomaterials. 35 (3), 916-928 (2014).</li><li>Cui, L. H., Joo, H. J., Kim, D. H., Seo, H. R., Kim, J. S., Choi, S. C., Huang, L. H., Na, J. E., Lim, I. R., Kim, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+anodic+aluminum+oxide:+anodization+and+templated+synthesis+of+functional+nanostructures.">Porous anodic aluminum oxide: anodization and templated synthesis of functional nanostructures.</a> Chemical reviews. 114 (15), 7487-7556 (2014).</li><li>Masuda, H., Fukuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+metal+nanohole+arrays+made+by+a+two-step+replication+of+honeycomb+structures+of+anodic+alumina.">Ordered metal nanohole arrays made by a two-step replication of honeycomb structures of anodic alumina.</a> science. 268 (5216), 1466 (1995).</li><li>Masuda, H., Satoh, M. <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+gold+nanodot+array+using+anodic+porous+alumina+as+an+evaporation+mask.">Fabrication of gold nanodot array using anodic porous alumina as an evaporation mask.</a> Japanese Journal of Applied Physics. 35 (1B), L126 (1996).</li><li>Zaraska, L., Sulka, G. D., Jasku&#322;a, M. <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+n-alcohols+on+porous+anodic+alumina+formed+by+self-organized+two-step+anodizing+of+aluminum+in+phosphoric+acid.">The effect of n-alcohols on porous anodic alumina formed by self-organized two-step anodizing of aluminum in phosphoric acid.</a> Surface and Coatings Technology. 204 (11), 1729-1737 (2010).</li><li>Yang, K. Y., Kim, J. W., Byeon, K. 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Fabbricazione di un AAO spesso soffre di difetti quali cricche, forme irregolari di pori e la masterizzazione. La ragione principale di questi difetti è chiamata un breakdown elettrolitico, che è fortemente influenzata dalla natura dei substrati metallici essendo anodizzato e la resistività del elettroliti21. Poiché la resistività dell'elettrolito varia a seconda della sua temperatura, eliminando calore continuamente dagli elettrodi è il punto critico per mantenere la temperatura tra piazze ...

Recentemente, la risposta delle cellule dalla stimolazione fisica della topografia superficiale è stato messo in luce nel campo della cella ingegneria1,2,3,4. Di conseguenza, si è concentrati più su nanostrutture tridimensionali al superficie cellulare allegato5. È stato segnalato che l'integrina, che è il dispositivo di riconoscimento superficie della cella, trasmette lo stimolo fisico guidato dalle strutture micro-nano di ECM a Meccano-trasduzione6. Questa stimolazione meccanica regola il comportamento delle cellule attraverso contatto Guida7 e induce la riorganizzazione del citoscheletro di cambiare forma, oltre alle adesioni focali e la rigidità delle cellule8.
Cellule progenitrici endoteliali umane (hEPCs) del corpo interagiscono strettamente con il microambiente circostante ECM9. Questo indica che lo stato fisico del ECM si comporta come un parametro importante per la formazione di complessi di adesione cellula-matrice specifica come shear stress derivato da sangue flusso10. È stato riferito che nanotopography superficie migliora la formazione in vitro di vasto tubo capillare reti di hEPCs11 e che un fattore solubile di ECM/bio sistema combinato consente hEPCs di riconoscere substrati disfunzionale e promuove Wound healing12,13. Tuttavia, il rapporto tra ECM e hEPCs non è chiaro.
Anche se molti ricercatori hanno cercato di chiarire il rapporto tra le risposte delle cellule e fisici spunti da diversi substrati14,15,16, questi studi utilizzati solo la dimensione fissa di una nanostruttura o nanopatterns con regime irregolare che hanno una limitazione per chiarire la relazione tra le dimensioni del comportamento delle cellule e nanostrutture. Il problema qui è una mancanza di idonei strumenti per lo screening delle risposte cellulari che possono sostituire approcci esistenti noiosi e iterativi per trovare la dimensione ottimale della nanostruttura. Di conseguenza, una tecnica semplice è richiesta per le reazioni di cella su stimolazioni fisiche senza ripetizione della selezione.
Qui, descriviamo un metodo utilizzato nella nostra precedente report17,18,19 per produrre un gradiente GNSM in cui il diametro della nanopillars organizzato aumenta progressivamente. Inoltre, abbiamo anche descritto come coltivare e analizzare il comportamento del hECFCs il gradiente GNSM piastre per determinare l'effetto di stimoli fisici sulle cellule. Un lieve anodizzazione, acquaforte graduale e metodo di rivestimento antiaderente strato sono stati utilizzati per fabbricare il gradiente della muffa AAO. Adottando una termica imprinting tecnica di Litografia, identico in polistirolo nanopatterns gradiente sono state prodotte in modo conveniente e facile. Utilizzando nanopatterns gradiente, è fattibile per determinare quale dimensione di nanostruttura ha un grande effetto sul comportamento delle cellule in un unico set di esperimento. Ci aspettiamo che questo gradiente GNSM sarà utile nella comprensione dei meccanismi di interazione tra hECFC derivate dal sangue o altre cellule e varie misure di nanostrutture.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Perchloric acid 60%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6512-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, absolute 99.9%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4118-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid 85%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6532-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>5558-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium(VI) oxide</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>2558-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>7781-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4104-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4081-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>8541-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>(heptadecafluoro-1,1,2,2,-tetrahydrodecyl)dimethylchlorosilane</td>
			<td>Gelest</td>
			<td>SIH5840.4</td>
			<td>Moisture sensitive</td>
		</tr>
		<tr>
			<td>Methoxynonafluorobutane 99%</td>
			<td>Sigma aldrich</td>
			<td>464309</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen solution</td>
			<td>Stemcell</td>
			<td>#4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma aldrich</td>
			<td>G1890</td>
			<td>Protein coating solution</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Heathcare</td>
			<td>17-1440-03</td>
			<td>Hydrophilic polysaccharide solution</td>
		</tr>
		<tr>
			<td>EGM-2MV</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Endothelial cell expansion medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma aldrich</td>
			<td>G5882-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma aldrich</td>
			<td>201030-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma aldrich</td>
			<td>X100-100ML</td>
			<td>Octylphenol ethoxylate&#160;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human vinculin primary antibody&#160;</td>
			<td>Sigma aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr>
			<td>F-actin probe</td>
			<td>Molecular Probes</td>
			<td>A12379</td>
			<td>Fluorescence-conjugated phalloidin</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated anti-mouse&#160;IgG antibody</td>
			<td>Molecular Probes</td>
			<td>A11001</td>
			<td>Fluorescence-conjugated secondary antibody&#160;</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole&#160;</td>
			<td>Sigma aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human vWF primary antibody&#160;</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human&#160;CD144 primary antibody&#160;</td>
			<td>BD Biosciences</td>
			<td>#555661</td>
			<td></td>
		</tr>
		<tr>
			<td>Eponate 12&#8482; Embedding Kit, with BDMA</td>
			<td>Ted Pella</td>
			<td>18012</td>
			<td>Epoxy resin</td>
		</tr>
		<tr>
			<td>Uranyl Acetate, 25g</td>
			<td>Ted Pella</td>
			<td>19481</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Citrate, Trihydrate, 10g</td>
			<td>Ted Pella</td>
			<td>19312</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure aluminum plate</td>
			<td>Goodfellow</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene sheet</td>
			<td>Goodfellow</td>
			<td>ST313120</td>
			<td></td>
		</tr>
		<tr>
			<td>8.0&#34; silicon wafer</td>
			<td>Siltron</td>
			<td>29-01024-03</td>
			<td>Single side polished, 725 &#181;m thick</td>
		</tr>
		<tr>
			<td>Vacuum desiccator, 4.4 L</td>
			<td>Kartell</td>
			<td>KA.230</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vacuumer</td>
			<td>V3.VOP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Unicorntech</td>
			<td>UDP-3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Daihan scientific</td>
			<td>SL.SMS03022</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>Daihan scientific</td>
			<td>HT120DX</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulator</td>
			<td>Daihan scientific</td>
			<td>WCR-P12</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear moving stage</td>
			<td>Zaber</td>
			<td>A-LSQ300A-E01-KT07</td>
			<td></td>
		</tr>
		<tr>
			<td>Angle bracket, 90 degrees</td>
			<td>Zaber</td>
			<td>AB90M</td>
			<td>Accessory of the linear moving stage</td>
		</tr>
		<tr>
			<td>PMP forcep, 145 mm</td>
			<td>Vitlab</td>
			<td>67995</td>
			<td>Nonmetallic tweezer</td>
		</tr>
		<tr>
			<td>PTFE beaker, 250 mL</td>
			<td>Cowie</td>
			<td>CW007.25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>B2510MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>PCB cutter</td>
			<td>Hozan Tool Industrial</td>
			<td>K-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoimprint device</td>
			<td>Nanonex</td>
			<td>NX-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma generator</td>
			<td>Femto Science</td>
			<td>CUTE</td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature sterilizer</td>
			<td>Lowtem</td>
			<td>Crystal 50</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Confoal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM6701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi</td>
			<td>H-7500</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of gradient nanopattern by thermal nanoimprinting technique and screening of the response of human endothelial colony-forming cells

Nanotopography può essere trovato in varie matrici extracellulari (ECMs) intorno al corpo ed è conosciuto per avere importanti atti normativi su reazioni cellulari. Tuttavia, è difficile da determinare la relazione tra le dimensioni di una nanostruttura e le risposte delle cellule a causa della mancanza di un'adeguata selezione di strumenti. Qui, ci mostra lo sviluppo delle piastre GNSM gradiente riproducibile ed economica per la manipolazione delle risposte cellulari. Utilizzando ossido di alluminio anodico (AAO) come uno stampo master, gradiente GNSM piastre con nanopillars delle gamme di diametro crescente [120-200 nm (GP 120/200), 200-280 nm (GP 200/280) e 280-360 nm (GP 280/360)] sono stati fabbricati da una termica imprinting tecnica. Queste piastre di gradiente GNSM sono state progettate per imitare le varie dimensioni dei nanotopography in ECM e sono state utilizzate per lo screening delle risposte delle cellule formanti colonie endoteliali umane (hECFCs). In questo protocollo, descriviamo la procedura dettagliata di fabbricare gradiente GNSM piastre per ingegneria, tecniche di coltivazione hECFCs da sangue periferico umano e hECFCs il GNSM piastre di coltura delle cellule.

La figura 1 Mostra immagini di SEM degli stampi AAO gradienti fabbricati secondo il loro tipo e la posizione. La figura 2 Mostra immagini di SEM di gradiente GNSM piastre con nanopillars regular-arrotondato, e nella figura 3 è dati di quantificazione del diametro nanopillars. La tabella 1 elenca le caratteristiche della nanopillars fabbricato.
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Watch this Scientific Journal Video about Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells at JoVE.com

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

Qui, presentiamo un protocollo per la fabbricazione di piastre gradiente GNSM via nanoimprinting termica e il metodo di screening risposte di cellule progenitrici endoteliali umane alle nanostrutture. Utilizzando la tecnologia descritta, è possibile produrre un ponteggio che può manipolare il comportamento delle cellule di stimoli fisici.

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

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

Research

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Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabbricazione di micro-tessuti utilizzando i moduli di Collagene Gel cellule contenenti

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

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Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

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Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

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Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

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Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

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Crescita degli strati cellulari endoteliali umani e ovini sulle membrane biomateriali

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

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Valutazione elettrofisiologia cardiaca preclinica mediante mappatura ottica a doppia tensione e calcio delle fette cardiache di organitypica umana

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The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...