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

Eric S. Hald; Kerianne E. Steucke; Jack A. Reeves; Zaw Win; Patrick W. Alford

doi:10.3791/52971

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Method Article

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

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DOI:

10.3791/52971

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June 26th, 2015

Eric S. Hald¹ , Kerianne E. Steucke¹ , Jack A. Reeves¹ , Zaw Win¹ , Patrick W. Alford¹

¹Department of Biomedical Engineering, University of Minnesota

Summary

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.

Abstract

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.

Introduction

Vascular diseases, such as cerebral vasospasm^1,2, hypertension³, and atherosclerosis⁴, develop slowly, are typically chronic in nature, and involve dysfunctional force-generation by vascular smooth muscle cells (VSMCs). We aim to study these slow-progressing vascular dysfunctions using in vitro methods with finer control of experimental conditions than in in vivo models. We have previously developed vascular muscular thin films (vMTFs) for measuring functional contractility of in vitro engineered cardiovascular tissues⁵, but this method has been limited to relatively short-term studies. Here, we pr....