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

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) [NRF-2015R1D1A1A01060397] and Bio &#38; Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT &#38; Future Planning [NRF-2017M3A9C6029563].

This study was approved by the Institutional Review Board at Korea University Anam Hospital (IRB No. ED170495). All procedures were carried out in accordance with the Helsinki Declaration and its later amendments.
1. Preparation of Aluminum (Al) Substrate by Electropolishing
Caution: Electropolishing solution is corrosive and toxic. Wear personal protective equipment including nitrile gloves, goggles and lab coat. Perform this step in a fume hood....

<ol>
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Fabrication of an AAO often suffers from defects such as cracks, irregular shapes of pores, and burning. The main reason for these defects is called an electrolytic breakdown, which is strongly affected by the nature of the metal substrates being anodized and the resistivity of the electrolyte21. Since the resistivity of the electrolyte varies depending on its temperature, eliminating heat continuously from electrodes is the critical point to maintain the locational temperature of the electrolyte ...

Recently, the response of cells by the physical stimulation of surface topography has been spotlighted in the field of cell engineering1,2,3,4. Therefore, more attention has been focused on three-dimensional nanostructures at the cell attachment surface5. It has been reported that the integrin, which is the surface recognition device of the cell, transmits the physical stimulus driven by the micro-nano structures of ECM through mechano-transduction6. This mechanical stimulation regulates cell behavior through contact guidance7 and induces cytoskeletal reorganization to change shape, in addition to focal adhesions and stiffness of cells8.
Human endothelial progenitor cells (hEPCs) in the body closely interact with the microenvironment of the surrounding ECM9. This indicates that the physical state of the ECM acts as an important parameter for specific cell-matrix adhesion complex formation as much as shear stress derived from blood flow10. It is reported that surface nanotopography enhances the in vitro formation of extensive capillary tube networks of hEPCs11 and that an ECM/bio soluble factor combined system enables hEPCs to recognize dysfunctional substrates and promotes wound healing12,13. Nonetheless, the relationship between ECM and hEPCs is not clearly understood.
Although many researchers tried to clarify the relationship between cell responses and physical cues from different substrates14,15,16, these studies used only the fixed size of a nanostructure or nanopatterns with irregular arrangements that have a limitation to elucidate the relationship between the size of the nanostructure and cell behavior. The problem here is a lack of suitable tools for screening cellular responses that can replace existing tedious and iterative approaches to find the optimum size of the nanostructure. Therefore, a straightforward technique is required for screening cell reactions on physical stimulations without repetition.
Here, we describe a method used in our previous reports17,18,19 to produce a gradient nanopattern in which the diameter of the arranged nanopillars gradually increases. In addition, we also described how to cultivate and analyze the behavior of hECFCs on gradient nanopattern plates to determine the effect of physical stimuli on the cells. A mild anodization, gradual etching, and anti-sticking layer coating method were used to fabricate gradient AAO mold. By adopting a thermal imprinting lithography technique, identical polystyrene gradient nanopatterns were produced in a cost-effective and facile way. Using gradient nanopatterns, it is feasible to determine which size of nanostructure has a great effect on cell behavior in one set of experiment. We expect that this gradient nanopattern will be helpful in understanding the interaction mechanisms between blood-derived hECFC or other cells and various sizes of nanostructures.

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			<td>Perchloric acid 60%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6512-4100</td>
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			<td>Ethyl alcohol, absolute 99.9%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4118-4100</td>
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			<td>Phosphoric acid 85%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6532-4400</td>
			<td></td>
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			<td>Methyl alcohol 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>5558-4400</td>
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			<td>Chromium(VI) oxide</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>2558-4400</td>
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			<td>Sulfuric acid 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>7781-4100</td>
			<td></td>
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			<td>Hydrogen peroxide 30%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4104-4400</td>
			<td></td>
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			<td>n-hexane 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4081-4400</td>
			<td></td>
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			<td>Toluene 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>8541-4400</td>
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			<td>(heptadecafluoro-1,1,2,2,-tetrahydrodecyl)dimethylchlorosilane</td>
			<td>Gelest</td>
			<td>SIH5840.4</td>
			<td>Moisture sensitive</td>
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			<td>Methoxynonafluorobutane 99%</td>
			<td>Sigma aldrich</td>
			<td>464309</td>
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			<td>Collagen solution</td>
			<td>Stemcell</td>
			<td>#4902</td>
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			<td>Gelatin</td>
			<td>Sigma aldrich</td>
			<td>G1890</td>
			<td>Protein coating solution</td>
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			<td>Ficoll-Paque</td>
			<td>GE Heathcare</td>
			<td>17-1440-03</td>
			<td>Hydrophilic polysaccharide solution</td>
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			<td>EGM-2MV</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Endothelial cell expansion medium</td>
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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
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			<td>Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>10010031</td>
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			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
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			<td>Paraformaldehyde</td>
			<td>Sigma aldrich</td>
			<td>P6148</td>
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			<td>Glutaraldehyde</td>
			<td>Sigma aldrich</td>
			<td>G5882-100ML</td>
			<td></td>
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			<td>Osmium tetroxide</td>
			<td>Sigma aldrich</td>
			<td>201030-1G</td>
			<td></td>
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			<td>Hexamethyldisilazane</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma aldrich</td>
			<td>X100-100ML</td>
			<td>Octylphenol ethoxylate&#160;</td>
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			<td>Goat serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
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			<td>anti-human vinculin primary antibody&#160;</td>
			<td>Sigma aldrich</td>
			<td>V9131</td>
			<td></td>
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			<td>F-actin probe</td>
			<td>Molecular Probes</td>
			<td>A12379</td>
			<td>Fluorescence-conjugated phalloidin</td>
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		<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>
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			<td>4&#39;,6-diamidino-2-phenylindole&#160;</td>
			<td>Sigma aldrich</td>
			<td>D9542</td>
			<td></td>
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			<td>Mounting medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
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			<td>Anti-human vWF primary antibody&#160;</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td></td>
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		<tr>
			<td>Anti-human&#160;CD144 primary antibody&#160;</td>
			<td>BD Biosciences</td>
			<td>#555661</td>
			<td></td>
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			<td>Eponate 12&#8482; Embedding Kit, with BDMA</td>
			<td>Ted Pella</td>
			<td>18012</td>
			<td>Epoxy resin</td>
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			<td>Uranyl Acetate, 25g</td>
			<td>Ted Pella</td>
			<td>19481</td>
			<td></td>
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		<tr>
			<td>Lead Citrate, Trihydrate, 10g</td>
			<td>Ted Pella</td>
			<td>19312</td>
			<td></td>
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			<td>Ultra pure aluminum plate</td>
			<td>Goodfellow</td>
			<td>26050-088</td>
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			<td>Polystyrene sheet</td>
			<td>Goodfellow</td>
			<td>ST313120</td>
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			<td>8.0&#34; silicon wafer</td>
			<td>Siltron</td>
			<td>29-01024-03</td>
			<td>Single side polished, 725 &#181;m thick</td>
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			<td>Vacuum desiccator, 4.4 L</td>
			<td>Kartell</td>
			<td>KA.230</td>
			<td></td>
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			<td>Vacuum pump</td>
			<td>Vacuumer</td>
			<td>V3.VOP100</td>
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			<td>Power supply</td>
			<td>Unicorntech</td>
			<td>UDP-3003</td>
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			<td>Magnetic stirrer</td>
			<td>Daihan scientific</td>
			<td>SL.SMS03022</td>
			<td></td>
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			<td>Overhead stirrer</td>
			<td>Daihan scientific</td>
			<td>HT120DX</td>
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		<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 can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Figure 1 shows SEM images of the fabricated gradient AAO molds according to their type and position. Figure 2 shows SEM images of gradient nanopattern plates with regular-rounded nanopillars, and Figure 3 is quantification data of the nanopillar diameter. Table 1 lists the characteristics of the fabricated 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

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

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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