title: A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues.
abstract: Engineered myocardial tissues can be used to elucidate fundamental features of myocardial biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged heart tissue in vivo. However, a key limitation is an inability to test the wide range of parameters (cell source, mechanical, soluble and electrical stimuli) that might impact the engineered tissue in a high-throughput manner and in an environment that mimics native heart tissue. Here we used microelectromechanical systems technology to generate arrays of cardiac microtissues (CMTs) embedded within three-dimensional micropatterned matrices. Microcantilevers simultaneously constrain CMT contraction and report forces generated by the CMTs in real time. We demonstrate the ability to routinely produce ~200 CMTs per million cardiac cells (<1 neonatal rat heart) whose spontaneous contraction frequency, duration, and forces can be tracked. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that both the dynamic force of cardiac contraction as well as the basal static tension within the CMT increased with boundary or matrix rigidity. Cell alignment is, however, reduced within a stiff collagen matrix; therefore, despite producing higher force, CMTs constructed from higher density collagen have a lower cross-sectional stress than those constructed from lower density collagen. We also study the effect of electrical stimulation on cell alignment and force generation within CMTs and we show that the combination of electrical stimulation and auxotonic load strongly improves both the structure and the function of the CMTs. Finally, we demonstrate the suitability of our technique for high-throughput monitoring of drug-induced changes in spontaneous frequency or contractility in CMTs as well as high-speed imaging of calcium dynamics using fluorescent dyes. Together, these results highlight the potential for this approach to quantitatively demonstrate the impact of physical parameters on the maturation, structure, and function of cardiac tissue and open the possibility to use high-throughput, low volume screening for studies on engineered myocardium.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Boudou
authors.forename: Wesley R
authors.initials: WR
authors.lastname: Legant
authors.forename: Anbin
authors.initials: A
authors.lastname: Mu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Borochin
authors.forename: Nimalan
authors.initials: N
authors.lastname: Thavandiran
authors.forename: Milica
authors.initials: M
authors.lastname: Radisic
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Epstein
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Margulies
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Chen
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006321:Heart / Q000502:physiology*; D055616:Microtechnology / Q000379:methods*; D009206:Myocardium / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods*
references.citation: Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18267-72
references.pmid: 19826086
references.citation: Am J Physiol. 1990 Apr;258(4 Pt 2):H977-82
references.pmid: 2158748
references.citation: Circ Res. 2005 Dec 9;97(12):1220-31
references.pmid: 16339494
references.citation: Cell Stem Cell. 2011 Feb 4;8(2):228-40
references.pmid: 21295278
references.citation: FASEB J. 1997 Jul;11(8):683-94
references.pmid: 9240969
references.citation: Circ Res. 2011 Feb 4;108(3):353-64
references.pmid: 21293007
references.citation: Cell Biochem Biophys. 2006;46(2):113-22
references.pmid: 17012753
references.citation: Cell. 2010 Aug 6;142(3):375-86
references.pmid: 20691899
references.citation: Biomaterials. 2002 Dec;23(24):4793-801
references.pmid: 12361618
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10097-102
references.pmid: 19541627
references.citation: J Thorac Cardiovasc Surg. 2002 Jul;124(1):63-9
references.pmid: 12091810
references.citation: Circulation. 1999 Nov 9;100(19 Suppl):II63-9
references.pmid: 10567280
references.citation: Circulation. 2000 Nov 7;102(19 Suppl 3):III56-61
references.pmid: 11082363
references.citation: Br J Pharmacol. 1993 Apr;108(4):1043-50
references.pmid: 8387382
references.citation: Circulation. 2006 Jul 4;114(1 Suppl):I72-8
references.pmid: 16820649
references.citation: Nature. 1985 May 2-8;315(6014):63-5
references.pmid: 2581143
references.citation: Nat Med. 2006 Apr;12(4):452-8
references.pmid: 16582915
references.citation: Am J Physiol. 1991 Mar;260(3 Pt 2):H884-92
references.pmid: 2000983
references.citation: Am J Physiol Heart Circ Physiol. 2006 Jun;290(6):H2196-203
references.pmid: 16473959
references.citation: Philos Trans R Soc Lond B Biol Sci. 2007 Aug 29;362(1484):1357-68
references.pmid: 17594967
references.citation: IEEE Trans Image Process. 2005 Sep;14(9):1372-83
references.pmid: 16190472
references.citation: J Pharmacol Exp Ther. 1970 May;173(1):145-51
references.pmid: 4245610
references.citation: Circ Res. 1991 Mar;68(3):836-46
references.pmid: 1742869
references.citation: Circ Res. 2002 Feb 22;90(3):e40
references.pmid: 11861428
references.citation: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1484-9
references.pmid: 12552122
references.citation: PLoS One. 2011 Apr 08;6(4):e18293
references.pmid: 21494607
references.citation: J Mol Cell Cardiol. 1991 Sep;23(9):1039-50
references.pmid: 1658347
references.citation: Acta Biomater. 2007 Jan;3(1):33-41
references.pmid: 17098488
references.citation: J Biomed Mater Res A. 2008 Sep;86(3):713-24
references.pmid: 18041719
references.citation: J Cell Sci. 2008 Nov 15;121(Pt 22):3794-802
references.pmid: 18957515
references.citation: Circ Res. 2009 Feb 27;104(4):e30-41
references.pmid: 19213953
references.citation: J Clin Invest. 2001 Aug;108(3):407-14
references.pmid: 11489934
references.citation: J Mol Cell Cardiol. 2011 Feb;50(2):320-6
references.pmid: 21040727
references.citation: Tissue Eng Part A. 2011 Jun;17(11-12):1465-77
references.pmid: 18783322
references.citation: Biotechnol Bioeng. 2003 May 20;82(4):403-14
references.pmid: 12632397
references.citation: Nat Protoc. 2009;4(2):155-73
references.pmid: 19180087
references.citation: J Pharmacol Exp Ther. 2003 Oct;307(1):419-28
references.pmid: 12954807
references.citation: Biotechnol Bioeng. 2000 Apr 5;68(1):106-14
references.pmid: 10699878
references.citation: Biotechnol Bioeng. 1999 Sep 5;64(5):580-9
references.pmid: 10404238
references.citation: Circ Res. 2002 Feb 8;90(2):223-30
references.pmid: 11834716
references.citation: Physiol Behav. 1991 Jul;50(1):47-52
references.pmid: 1946730
references.citation: FASEB J. 2000 Apr;14(5):669-79
references.pmid: 10744624
references.citation: Circ Res. 2010 Jul 9;107(1):35-44
references.pmid: 20448218
references.citation: Curr Opin Crit Care. 2002 Oct;8(5):395-403
references.pmid: 12357106
references.citation: Eur J Pharmacol. 1996 Dec 30;318(2-3):349-56
references.pmid: 9016925
references.citation: Proc Natl Acad Sci U S A. 2004 Dec 28;101(52):18129-34
references.pmid: 15604141
references.citation: Circulation. 2004 Aug 24;110(8):962-8
references.pmid: 15302801
references.citation: In Vitro Cell Dev Biol. 1990 Sep;26(9):905-14
references.pmid: 2172205
references.citation: Biomaterials. 2011 Feb;32(4):1002-9
references.pmid: 21071078
references.citation: Biotechnol Bioeng. 2009 Feb 1;102(2):493-507
references.pmid: 18767184