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. 2012 May;18(9-10):910-9.
doi: 10.1089/ten.tea.2011.0341. Epub 2012 Jan 4.

A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues

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A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues

Thomas Boudou et al. Tissue Eng Part A. 2012 May.

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.

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Figures

FIG. 1.
FIG. 1.
Temporal evolution of CMTs constructed in 0.5 mg/mL fibrin and 1.0 mg/mL collagen gels and tethered to rigid (k=0.45 μN/μm) cantilevers. (A) Representative images depicting the time course of a contracting CMT. (B) Representative recording of the tension as a function of the time of a CMT on day 5. (C) Temporal evolution of the spontaneous beating frequency and (D) of the contraction duration of beating (contraction and relaxation). Data are the average of 15 CMTs±SEM. *p<0.01. CMT, cardiac microtissue.
FIG. 2.
FIG. 2.
Influence of the pillar stiffness on dynamic and static contractility of CMTs constructed in 0.5 mg/mL fibrin and 1.0 mg/mL collagen gels. (A) Average tension, (B) cross-sectional area, and (C) cross-sectional stress for CMTs tethered to flexible (k=0.20 μN/μm) or rigid (k=0.45 μN/μm) cantilevers at day 5. Data are the average of 15 CMTs±SEM. (D) Corresponding phase-contrast images. *p<0.01; **p<0.01.
FIG. 3.
FIG. 3.
Influence of the matrix composition on dynamic and static contractility of CMTs tethered to rigid (k=0.45 μN/μm) cantilevers. (A) Average tension, (B) cross-sectional area, and (C) cross-sectional stress for CMTs constructed from 0.5 mg/mL fibrin and 1.0 mg/mL or 2.5 mg/mL collagen gels at day 5. Data are the average of 15 CMTs±SEM. (D) Corresponding phase-contrast images. *p<0.01, **p<0.01.
FIG. 4.
FIG. 4.
Functional properties over days of nonstimulated and electrically stimulated CMTs constructed in 0.5 mg/mL fibrin and 1.0 mg/mL collagen gels and tethered to rigid (k=0.45 μN/μm) cantilevers. (A) Representative recording of the tension in function of the time and the pacing rate of a nonstimulated CMT on day 5. (B) Maximum capture rate. (C) Voltage threshold. (D) Static and dynamic cross-sectional stress. Data from (B–D) are the average of 10 CMTs±SEM. *p<0.01. Color images available online at www.liebertonline.com/tea
FIG. 5.
FIG. 5.
Structural properties and calcium signaling of CMTs constructed in 0.5 mg/mL fibrin and 1.0 mg/mL collagen gels and tethered to rigid (k=0.45 μN/μm) cantilevers. (A, B) Immunostaining of troponin-T (red) and nuclei (blue) in a representative CMT at day 3 and (C, D) at day 7. (E) Percentage of aligned nuclei (angle<20°). (F) Time series fluorescence images of a CMT after incubation with calcium indicator fluo-3. (G) Representative simultaneous recording of the fluorescence of the calcium indicator and the tension exerted by a CMT. Data from (E) are the average of 6 CMTs±SEM. Color images available online at www.liebertonline.com/tea
FIG. 6.
FIG. 6.
Effects of isoproterenol and digoxin on CMT functionality. (A) Variation of the dynamic cross-sectional stress compared with the control and (B) of the beating frequency in function of the drug concentration. Data are the average of 10 CMTs±SEM. *p<0.01; #p<0.01.

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