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

Dette arbejde blev støttet af den grundlæggende videnskab forskningsprogram gennem National Research Foundation af Korea (NRF) finansieret af Ministeriet for uddannelse, videnskab og teknologi (MEST) [NRF-2015R1D1A1A01060397] og Bio & medicinsk teknologiudvikling Program af NRF finansieret af Ministeriet for videnskab, IKT & fremtid planlægning [NRF-2017M3A9C6029563].

Denne undersøgelse blev godkendt af det institutionelle Review Board på Korea Anam Universitetshospital (IRB nr. ED170495). Alle procedurer blev gennemført i overensstemmelse med Helsinki-erklæringen med senere ændringer.
1. fremstilling af aluminium (Al) substrat af Electropolishing
Forsigtighed: Electropolishing løsning er ætsende og giftige. Bære personlige værnemidler, herunder nitrilhandsker, briller og lab coat. Udføre dette trin i et ...

<ol>
	<li>Dalby, M. J., Gadegaard, N., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+nanotopography+and+integrin-matrix+interactions+to+influence+stem+cell+fate.">Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate.</a> Nature materials. 13 (6), 558-569 (2014).</li><li>Qian, W., Gong, L., Cui, X., Zhang, Z., Bajpai, A., Liu, C., Castillo, A., Teo, J. C., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographic+Regulation+of+Human+Mesenchymal+Stem+Cell+Osteogenesis.">Nanotopographic Regulation of Human Mesenchymal Stem Cell Osteogenesis.</a> ACS Applied Materials &amp; Interfaces. 9 (48), 41794-41806 (2017).</li><li>Naganuma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+cell+adhesion+force+activation+on+nano/micro-topographical+surfaces+and+temporal+dependence+of+cell+morphology.">The relationship between cell adhesion force activation on nano/micro-topographical surfaces and temporal dependence of cell morphology.</a> Nanoscale. 9 (35), 13171-13186 (2017).</li><li>Han, J., Lin, K. H., Chew, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+regulation+of+focal+adesions+and+cortical+actin+by+matrix+nanotopography+in+3D+environment.">Study on the regulation of focal adesions and cortical actin by matrix nanotopography in 3D environment.</a> Journal of Physics: Condensed Matter. 29 (45), 455101 (2017).</li><li>Liu, X., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+nano-biointerface+as+a+new+platform+for+guiding+cell+fate.">Three-dimensional nano-biointerface as a new platform for guiding cell fate.</a> Chemical Society Reviews. 43 (8), 2385-2401 (2014).</li><li>Turner, L. A., Dalby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopography-potential+relevance+in+the+stem+cell+niche.">Nanotopography-potential relevance in the stem cell niche.</a> Biomaterials science. 2 (11), 1574-1594 (2014).</li><li>Driscoll, M. K., Sun, X., Guven, C., Fourkas, J. T., Losert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+contact+guidance+through+dynamic+sensing+of+nanotopography.">Cellular contact guidance through dynamic sensing of nanotopography.</a> ACS nano. 8 (4), 3546-3555 (2014).</li><li>Yim, E. K., Darling, E. M., Kulangara, K., Guilak, F., 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=Nanotopography-induced+changes+in+focal+adhesions,+cytoskeletal+organization,+and+mechanical+properties+of+human+mesenchymal+stem+cells.">Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.</a> Biomaterials. 31 (6), 1299-1306 (2010).</li><li>Davis, G. E., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+extracellular+matrix.">Endothelial extracellular matrix.</a> Circulation research. 97 (11), 1093-1107 (2005).</li><li>Nakayama, K. H., Surya, V. N., Gole, M., Walker, T. W., Yang, W., Lai, E. S., Ostrowski, M. A., Fuller, G. G., Dunn, A. R., Huang, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+patterning+of+extracellular+matrix+alters+endothelial+function+under+shear+stress.">Nanoscale patterning of extracellular matrix alters endothelial function under shear stress.</a> Nano letters. 16 (1), 410-419 (2015).</li><li>Bettinger, C. J., Zhang, Z., Gerecht, S., Borenstein, J. T., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+in+vitro+capillary+tube+formation+by+substrate+nanotopography.">Enhancement of in vitro capillary tube formation by substrate nanotopography.</a> Advanced materials. 20 (1), 99-103 (2008).</li><li>Katz, B. Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction.">Endothelial function and dysfunction.</a> Circulation. 115 (10), 1285-1295 (2007).</li><li>Tajima, S., Chu, J., Li, S., Komvopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+endothelial+cell+adhesion,+spreading,+and+cytoskeleton+on+low-density+polyethylene+by+nanotopography+and+surface+chemistry+modification+induced+by+argon+plasma+treatment.">Differential regulation of endothelial cell adhesion, spreading, and cytoskeleton on low-density polyethylene by nanotopography and surface chemistry modification induced by argon plasma treatment.</a> Journal of Biomedical Materials Research Part A. 84 (3), 828-836 (2008).</li><li>Mohiuddin, M., Pan, H. A., Hung, Y. C., Huang, G. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopillar+Surface+Topology+Promotes+Cardiomyocyte+Differentiation+through+Cofilin-Mediated+Cytoskeleton+Rearrangement.">Nanopillar Surface Topology Promotes Cardiomyocyte Differentiation through Cofilin-Mediated Cytoskeleton Rearrangement.</a> ACS Applied Materials &amp; Interfaces. 9 (20), 16803-16812 (2017).</li><li>Hwang, J. H., Lee, D. H., Byun, M. R., Kim, A. R., Kim, K. M., Park, J. I., Oh, H. T., Hwang, E. S., Lee, K. B., Hong, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopological+plate+stimulates+osteogenic+differentiation+through+TAZ+activation.">Nanotopological plate stimulates osteogenic differentiation through TAZ activation.</a> Scientific Reports. 7 (1), 3632 (2017).</li><li>Bae, D., Moon, S. H., Park, B. G., Park, S. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+the+response+of+human+endothelial+colony-forming+cells+by+focal+adhesion+assembly+using+gradient+nanopattern+plates.">Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.</a> Acta biomaterialia. 65, 272-282 (2017).</li><li>Lee, W., Park, S. 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. J., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+deposition+of+the+silver+nano-particles+using+patterned+the+hydrophobic+self-assembled+monolayer+patterns+and+zero-residual+nano-imprint+lithography.">Selective deposition of the silver nano-particles using patterned the hydrophobic self-assembled monolayer patterns and zero-residual nano-imprint lithography.</a> Microelectronic engineering. 84 (5), 1552-1555 (2007).</li><li>Park, B. G., Lee, W., Kim, J. S., Lee, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superhydrophobic+fabrication+of+anodic+aluminum+oxide+with+durable+and+pitch-controlled+nanostructure.">Superhydrophobic fabrication of anodic aluminum oxide with durable and pitch-controlled nanostructure.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 370 (1), 15-19 (2010).</li></ol>

Fabrikation af en AAO ofte lider af mangler såsom revner, uregelmæssige former af porer, og afbrænding. Den væsentligste årsag til disse fejl kaldes en elektrolytisk opdeling, der er stærkt påvirket af arten af de metal substrater er anodiseret, resistivitet elektrolyt21. Da resistivitet af elektrolytten varierer afhængigt af dens temperatur, er fjerne varmen kontinuerligt fra elektroder det kritiske punkt til at opretholde de placeringsmæssige temperatur af elektrolytten stabil i sådan ...

For nylig har været spotlighted reaktion af celler ved fysisk stimulering af overflade topografi inden for celle engineering1,2,3,4. Derfor, mere opmærksomhed været rettet på tre-dimensionelle nanostrukturer på celle vedhæftet fil overflade5. Det er blevet rapporteret, at Integra, som er enhedens overflade anerkendelse af cellen, sender den fysiske stimulus drevet af mikro-nano strukturer af ECM gennem mekanisk-transduktion6. Denne mekaniske stimulation regulerer celle adfærd gennem kontakt vejledning7 og inducerer cytoskeletal reorganisering til at ændre form, fokale sammenvoksninger og stivhed af celler8.
Menneskelige endotelceller stamceller (hEPCs) i kroppen interagere tæt med mikromiljø af omkringliggende ECM-9. Dette indikerer, at den fysiske tilstand af ECM fungerer som en vigtig parameter for specifikke celle-matrix vedhæftning kompleks dannelse som shear stress afledt af blod flow10. Det forlyder at overflade nanotopography forbedrer in vitro- dannelsen af omfattende kapillarrør netværk af hEPCs11 , og at en ECM/bio opløselige faktor kombineret system gør det muligt for hEPCs at genkende dysfunktionelle substrater og fremmer sår healing12,13. Ikke desto mindre, forholdet mellem ECM og hEPCs er ikke klart forstået.
Selv om mange forskere forsøgt at afklare forholdet mellem celle svar og fysiske signaler fra forskellige substrater14,15,16, disse undersøgelser bruges kun den faste størrelse med en nanostrukturer eller nanopatterns med irregulære ordninger, der har en begrænsning til at belyse forholdet mellem størrelsen af nanostrukturer og celle adfærd. Problemet her er mangel på egnede værktøjer til screening cellulære svar, der kan erstatte eksisterende kedelig og iterative fremgangsmåder for at finde den optimale størrelse af nanostrukturer. En enkel teknik er derfor påkrævet for screening celle reaktioner på fysisk stimulering uden gentagelse.
Her, beskriver vi en metode, der anvendes i vores tidligere rapporter17,18,19 til at producere en gradient nanopattern som diameteren af de arrangerede nanopillars gradvist øges. Derudover beskrevet vi også, hvordan man dyrker og analysere funktionen af hECFCs på gradient nanopattern plader til at bestemme virkningen af fysiske stimuli på cellerne. En mild anodization, gradvis ætsning og anti-stikning lag belægning metode blev brugt til at fabrikere gradient AAO skimmel. Ved at vedtage en termisk prægning litografi teknik, var identiske polystyren gradient nanopatterns produceres på en omkostningseffektiv og letkøbt måde. Ved hjælp af gradient nanopatterns, er det muligt at afgøre, hvilken størrelse af nanostrukturer har en stor effekt på celle opførsel i ét sæt af eksperiment. Vi forventer, at denne gradient nanopattern vil være nyttige i forståelse samspil mekanismer mellem blod-afledte hECFC eller andre celler og forskellige størrelser af nanostrukturer.

<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 kan findes i forskellige ekstracellulære matricer (ECMs) rundt i kroppen og er kendt for at have vigtige lovgivningsmæssige aktioner på cellulære reaktioner. Det er imidlertid vanskeligt at bestemme forholdet mellem størrelsen af en nanostrukturer og svar af celler på grund af manglen på ordentlig screening-værktøjer. Her viser vi udviklingen af reproducerbar og omkostningseffektiv gradient nanopattern plader til manipulation af cellulære svar. Ved hjælp af anodisk aluminiumoxid (AAO) som en master mold, gradient nanopattern plader med nanopillars af stigende diameter intervaller [120-200 nm (GP 120/200), 200-280 nm (GP 200/280) og 280-360 nm (GP 280/360)] blev fremstillet af en termisk prægning teknik. Disse gradient nanopattern plader var designet til at efterligne de forskellige størrelser af nanotopography i ECM og blev anvendt til at screene svar af menneskelige endotel kolonidannende celler (hECFCs). I denne protokol beskrive vi den trinvise procedure af opdigte gradient nanopattern plader til celle teknikker, dyrke hECFCs fra humant perifert blod, og dyrkning af hECFCs på nanopattern plader.

Figur 1 viser SEM billeder af de fabrikerede gradient AAO forme efter deres type og beliggenhed. Figur 2 viser SEM billeder af gradient nanopattern plader med regelmæssig afrundede nanopillars, og figur 3 er kvantificering data af nanopillar diameter. Tabel 1 viser de særlige kendetegn ved den fabrikerede nanopillars.
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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

Vi præsenterer her, en protokol for fabrikation af gradient nanopattern plader via termisk nanoimprinting og metoden til screening svar af menneskelige endotelceller stamceller til nanostrukturer. Ved hjælp af den beskrevne teknologi, er det muligt at producere et stillads, der kan manipuleres celle adfærd ved fysiske stimuli.

Fabrikation af Gradient Nanopattern af termisk Nanoimprinting teknik og Screening af svar i Endothelial kolonidannende celler

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

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