We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

<ol>
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(Camb). 3, 1063-1070 (2011).</li><li>Win, Z., 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=Smooth+muscle+architecture+within+cell-dense+vascular+tissues+influences+functional+contractility).">Smooth muscle architecture within cell-dense vascular tissues influences functional contractility).</a> Integr. Biol. (Camb). , (2014).</li><li>Genchi, G. G., 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=Bio/non-bio+interfaces:+a+straightforward+method+for+obtaining+long+term+PDMS/muscle+cell+biohybrid+constructs).">Bio/non-bio interfaces: a straightforward method for obtaining long term PDMS/muscle cell biohybrid constructs).</a> Colloid Surface B. 105, 144-151 (2013).</li><li>Fessel, G., Cadby, J., Wunderli, S., van Weeren, R., Snedeker, J. 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The authors have nothing to disclose.

A continuación, presentamos un protocolo que se basa en la tecnología vMTF desarrollado previamente, lo que permite tiempos de experimentación extendidos más típica de las vías de enfermedades vasculares crónicas 1,23,24. Para lograr esto, micropatrón genipina, que ha sido previamente demostrado para proporcionar funcionalización a largo plazo de sustratos PDMS 11, utilizando una técnica de deposición de microfluidos para producir laminillas arterial Engineered con la mejora de la viabil...

Las enfermedades vasculares, tales como vasoespasmo cerebral 1,2, hipertensión 3, 4 y la aterosclerosis, se desarrollan lentamente, son típicamente de naturaleza crónica, e implican disfuncional fuerza generación por las células del músculo liso vascular (CMLV). Nuestro objetivo es estudiar estos lento que progresan disfunciones vasculares utilizando métodos in vitro con un mayor control de las condiciones experimentales que en modelos in vivo. Hemos desarrollado previamente películas finas musculares vasculares (vMTFs) para la medición de la contractilidad funcional de in vitro diseñados tejidos cardiovasculares 5, pero este método se ha limitado a relativamente estudios a corto plazo. A continuación, presentamos una técnica de modificación del sustrato que se expande nuestra técnica anterior vMTF para mediciones a largo plazo. Mientras que el endotelio es también crítico en la función vascular en general, laminillas arterial ingeniería proporcionan un sistema modelo útil para evaluar los cambios en vascularcontractilidad durante la progresión de la enfermedad. Para diseñar un modelo de tejido de la enfermedad vascular funcional, tanto la estructura y la función de la lámina arterial, la unidad contráctil básico de la embarcación, se debe recapitula con alta fidelidad. Laminillas arterial son láminas concéntricas, circunferencialmente alineados de CMLV contráctiles separadas por láminas de elastina 6. La impresión por microcontacto de (ECM), las proteínas de la matriz extracelular sobre sustratos de polidimetilsiloxano (PDMS) se ha utilizado anteriormente para proporcionar señales de orientación para la organización del tejido para imitar alineado tejido cardiovascular 5,7-10. Sin embargo, los tejidos modelados utilizando impresión por microcontacto puede perder la integridad después de 3-4 días de cultivo, lo que limita su aplicabilidad en estudios crónicos. Este protocolo proporciona una solución a este problema mediante la sustitución de las técnicas de impresión por microcontacto anteriores con una nueva técnica de deposición de microfluidos. Genchi et al. PDMS sustratos modificados con genipina y found prolongada viabilidad de miocitos hasta un mes en la cultura 11. Aquí, se utiliza un enfoque similar para extender la cultura de las células del músculo liso vascular estampadas en PDMS. Genipina, un derivado hidrolítica natural de la fruta gardenia, es un candidato deseable para la modificación de sustrato debido a su toxicidad relativamente baja en comparación con agentes de reticulación similares y su creciente uso como un biomaterial en los campos de la reparación de tejidos y la modificación 12,13 ECM 14, 15. En este protocolo, la fibronectina se utiliza como una señal de orientación celular, como en métodos de impresión por microcontacto anteriores; sin embargo, genipina se deposita sobre PDMS sustratos antes de patrón fibronectina. Por lo tanto, como las células degradan la matriz de estampado, ECM recién sintetizado a partir de CMLV adjuntos se puede unir al sustrato PDMS genipina-revestido. Este protocolo utiliza un dispositivo de suministro de microfluidos para el de dos pasos genipina y ECM deposición. El diseño de la microfluidos imita dispositivo microcopatrones de impresión ntact utilizados para laminillas arterial ingeniería en estudios previos 16. Por lo tanto, esperamos que este protocolo para dar imita laminillas arterial que recapitular con éxito el alineado altamente en la estructura y la función contráctil vivo de laminillas arterial. También evaluamos la contractilidad del tejido que confirmar que genipina es un compuesto de modificación sustrato adecuado para largo plazo en modelos de enfermedades vasculares in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Coverslip staining rack</td>
			<td>Electron Microscopy Sciences</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com/</a></td>
			<td>72239-04</td>
			<td>Alternative coverslip rack may be used</td>
		</tr>
		<tr>
			<td>Microscope cover glass - 25 mm</td>
			<td>Fisher Scientific, Inc.</td>
			<td><a href="http://www.fishersci.com/">www.fishersci.com</a></td>
			<td>12-545-102</td>
			<td>Alternative brand and size may be used; Microscope slides may also be substituted as substrate base</td>
		</tr>
		<tr>
			<td>Poly(N-iso-propylacrylamide) (PIPAAm)</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td>#21458</td>
			<td>Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation</td>
		</tr>
		<tr>
			<td>1-butanol</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td align="right">360465</td>
			<td>Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 &#176;C, avoid heating; alternative product may be used</td>
		</tr>
		<tr>
			<td>Spincoater</td>
			<td>Specialty Coating Systems, Inc.</td>
			<td><a href="http://www.scscoatings.com/">www.scscoatings.com</a></td>
			<td></td>
			<td>SCS G3P8 Model; Alternative brand and/or model may be used</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives (Dow Corning)</td>
			<td><a href="http://www.ellsworth.com/">www.ellsworth.com</a></td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Alternative distributor may be used</td>
		</tr>
		<tr>
			<td>Fluorescent microbeads</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td align="right">17151</td>
			<td>Alternative brand and/or larger size may be used</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Wafer World, Inc.</td>
			<td><a href="http://www.waferworld.com/">www.waferworld.com</a></td>
			<td align="right">2398</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Photoresist&#160;</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 3025 allows 20-25-&#181;m feature height</td>
		</tr>
		<tr>
			<td>Contact mask aligner</td>
			<td>Suss MicroTec</td>
			<td><a href="http://www.suss.com/">www.suss.com</a></td>
			<td></td>
			<td>MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure</td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 Developer; Hazard: flammable</td>
		</tr>
		<tr>
			<td>Tridecafluro-trichlorosilane</td>
			<td>UCT Specialties, Inc.</td>
			<td><a href="http://www.unitedchem.com/">www.unitedchem.com</a></td>
			<td>T2492</td>
			<td>Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. )</td>
		</tr>
		<tr>
			<td>Surgical biopsy punch</td>
			<td>Integra LifeSciences Corp.</td>
			<td><a href="http://www.miltex.com/">www.miltex.com</a></td>
			<td>33-31AA-P/25</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Cayman Chemical</td>
			<td><a href="http://www.caymanchem.com/">www.caymanchem.com</a></td>
			<td align="right">10010622</td>
			<td>Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>Mediatech, Inc.</td>
			<td><a href="http://www.cellgro.com/">www.cellgro.com</a></td>
			<td>21-031-CV</td>
			<td>Alternative brand may be used</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning, Inc.</td>
			<td><a href="http://www.corning.com/">www.corning.com</a></td>
			<td align="right">356008</td>
			<td>Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Inc.</td>
			<td><a href="http://www.lifetechnologies.com/">www.lifetechnologies.com</a></td>
			<td>15140-122</td>
			<td>Alternative brand and/or size may be used, as long as concentration is the same</td>
		</tr>
		<tr>
			<td>Umbillical artery smooth muscle cells</td>
			<td>Lonza</td>
			<td><a href="http://www.lonza.com/">www.lonza.com</a></td>
			<td>CC-2579</td>
			<td>Alternative cell types may be used for alternative applications. Media should be modified accordingly</td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution components</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>various</td>
			<td>Alternative brand may be used for mixing solution</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
			<td>4350020000000000</td>
			<td>SteREOLumar V12; Alternative brand/type of stereomicroscope may be used</td>
		</tr>
		<tr>
			<td>Temperature-controlled platform</td>
			<td>Warner Instruments</td>
			<td><a href="http://www.warneronline.com/">www.warneronline.com</a></td>
			<td>641659; 640352; 641922</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>E7764-50UG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
		<tr>
			<td>HA-1077</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>H139-10MG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
	</tbody>
</table>

microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

El objetivo principal de este trabajo fue extender la viabilidad de CMLV micropatterned sobre sustratos PDMS hidrofóbicas. Esto se logró mediante la incorporación de un sistema de entrega de microfluidos para depositar genipina modelado y fibronectina en PDMS (Figura 1). La deposición de proteínas ECM utilizando la entrega de microfluidos dio de alta transferencia de la fidelidad de la pauta canal con PDMS desnudas entre líneas de genipina y fibronectina (Figura 1D). Las células ...

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Microfluidic genipina Deposición Técnica de Cultura extendido de micropatterned vasculares musculares Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

microfluidic-genipin-deposition-technique-for-extended-culture

Research

JoVE Journal

Bioengineering

7.6K vistas.  University of Minnesota. We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.