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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Capillary Force Lithography for Cardiac Tissue Engineering

Published: June 10th, 2014

DOI:

10.3791/50039

1Department of Bioengineering, University of Washington, 2Department of Pathology, University of Washington

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 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’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 (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °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.

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 wall

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All procedures are conducted at room temperature (~23 °C) unless otherwise noted.

1. Fabrication of Silicon Master

  1. Clean silicon wafer with 100% ethanol or xylene and dry under O2/N2 gas.
  2. Place silicon wafer in spin-coater at rotation speeds of 2,000-4,000 rpm to produce a 0.3-0.5 µm thick film.
  3. Pattern the photoresist film with the correct dimensions by using a photolithography system
  4. Fully immerse the patterned photore.......

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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.......

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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.......

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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.  Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

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Name Company Catalog Number Comments
Name Company Catalogue number Comments (optional)
Fibronectin BD Biosciences 354008
NOA 76 Norland Products, Inc. 7606B
Surface Adhesion Promotor (Glass Primer) Minuta Tech
PUA Minuta Tech MINS-311RM
Soft Rubber Roller Speedball
Silicon Wafers NOVA Electronic Materials FA01-9900
Photoresist Shipley SPRT510
Photoresist Developer Shipley MF320
Electron-Beam Lithography System JEOL JBX-9300FS
Etching System Surface Technology Systems NP10 8UJ
Plasma Asher System BMR Technology Co. DSF-200
Ozone Cure System Minuta Tech MT-UV-O- 08
Fusion Cure System Minuta Tech MT-UV-A 11
NOA 83H Norland Products, Inc. 8301
Spin Coater Laurel Technology WS-400-6NPP
Skyrol PET Film SKC Co., Ltd. 23038-59-9
25mm Glass Slides Corning 2948
Sylgard 184 Silicone Elastomer Kit Dow Corning 6/5/2553
Poly(D,L-lactide-co-glycolide) Sigma-Aldrich P2191-1G
Chloroform Sigma-Aldrich 372978-1L
500g Weights Global Insustrial T9FB503120
Isopropyl Alcohol EMD Millipore PX1835-2
Hot Plate Corning PC-420D
Sonicator Branson B2510MTH

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