Name: capillary force lithography for cardiac tissue engineering
Uploaded: 2014-06-10T13:00:00
Description: Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

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.

All procedures are conducted at room temperature (~23 &#176;C) unless otherwise noted.
1. Fabrication of Silicon Master
<ol>
	<li>Clean silicon wafer with 100% ethanol or xylene and dry under O2/N2 gas.</li>
	<li>Place silicon wafer in spin-coater at rotation speeds of 2,000-4,000 rpm to produce a 0.3-0.5 &#181;m thick film.</li>
	<li>Pattern the photoresist film with the correct dimensions by using a photolithography system</li>
	<li>Fully immerse the patterned photore...

<ol>
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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. -. 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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. 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Functionally mature cardiac tissues are lacking for both in vivo and in vitro applications of cardiac tissue engineering. The CFL nanofabrication methods described here are robust techniques for achieving cellular alignment and influencing macroscopic tissue function due to the scalability of the system. Large areas can easily be patterned and used for cell culture. Macroscopic cellular alignment is essential in cardiac tissue engineering in order to create biomimetic, functional tissue as it influences...

Cardiovascular disease is the leading cause of morbidity and mortality in the world and present a weighty socio-economic burden on an already strained global health system1,17. Cardiac tissue engineering has two distinct goals: (1) to regenerate damaged myocardium after ischemic disease or cardiomyopathy or (2) to create a high fidelity model of the heart for in vitro drug screening or disease modeling.
The heart is a complex organ that must work constantly to supply blood to the body. Densely packed laminar structures of cardiomyocytes and supportive tissues are arranged in helical patterns throughout the heart wall18,19. The heart is also electromechanically coupled20 in a highly coordinated fashion to efficiently eject blood to the body21. Several major hurdles remain to be addressed, however, before nature&#8217;s intricate design can reliably be recapitulated in vitro. First, although robust cardiomyocyte differentiation methods continue to be developed22, hPSC-CMs still exhibit rather immature phenotypes. Their electromechanical properties and morphology most closely match fetal levels23. Second, when kept in traditional culture conditions, both stem cell-derived and primary cardiomyocytes fail to assemble into native, tissue-like structures. Rather, cells become randomly oriented and do not exhibit the banded rod-shaped appearance of adult myocardium24.
The extracellular matrix (ECM) environment with which cells interact plays a significant role in numerous cellular processes11,13,25. The ECM consists of complex, well-defined molecular and topographical cues that significantly influence the structure and function of cells6,26. Within the heart, cellular alignment closely follows the underlying nanometer scale ECM fibers2. The impact of these nanotopographical cues on cell and tissue function, however, is far from completely understood. Preliminary studies of nanometer scale cell-biomaterial interaction indicate the potential importance and impact of sub-micron topographical cues for cell signaling27, adhesion28-30, growth31, and differentiation32,33. However, due to the difficulty in developing reproducible and scalable nanofabricated substrates, such studies could not reproduce the multi-scale cellular effects of the complex in vivo ECM environment. In this protocol, a straightforward and cost-effective nanofabrication technique to produce cell culture scaffolds mimicking native cardiac ECM fiber alignment is described, allowing for a wide range of novel investigations of cardiomyocyte-biomaterial interactions. Understanding how cardiomyocytes interact with the nanoscale ECM environment could allow for the ability to control cellular behavior to more closely mimic native tissue function. Furthermore, cell monolayers are a simplified experimental system compared to 3D structures but still exhibit complex multi-cellular behavior for insightful investigations and functional screening2,34-36. Finally, such scaffolds could be used to improve cellular graft function when implanted into the heart for regenerative purposes37.

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		<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>
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		<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

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

Figure 1 is a schematic overview of the production process for the two fabrication methods. Due to the diffraction of light caused by the nanoscale topography, nanopatterning should result in an iridescent surface to the ANFS. Figure 2 depicts this iridescent surface on a well-patterned 25 mm NP-PU coverslip (Figure 2A) with 800 nm ridge and groove width (Figure 2B). The iridescent appearance of the ANFS will vary slightly depending on the ridge and groo...

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.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Research

JoVE Journal

Bioengineering

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Plasma Lithography Surface Patterning for Creation of Cell Networks

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Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

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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 ...

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

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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. ...

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 ...

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

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 ...

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 ...