Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Tissue Eng Part A. 2012 May;18(9-10):910-9. doi: 10.1089/ten.tea.2011.0341. Epub 2012 Jan 4.
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.
工程化心肌组织可用于阐明心肌生物学的基本特征、开发器官型体外模型系统，以及作为工程化组织构建物在体内替代受损的心脏组织。然而，一个关键的限制是无法以高通量的方式并在模拟天然心脏组织的环境中测试可能影响工程化组织的广泛参数（细胞来源、机械、可溶性和电刺激）。在这里，我们使用微机电系统技术生成嵌入在三维微图案基质中的心脏微组织（CMT）阵列。微悬臂梁同时约束 CMT 的收缩，并实时报告 CMT 产生的力。我们证明了能够常规地生产每百万个心脏细胞 (~200 个 CMT)，每个 CMT 的自发收缩频率、持续时间和力都可以被跟踪。独立地改变微悬臂梁和胶原基质的机械刚度，结果表明，心脏收缩的动态力以及 CMT 内的基础静态张力都随边界或基质的刚性而增加。然而，在刚性胶原基质中，细胞排列会减少；因此，尽管产生更高的力，但由高密度胶原构建的 CMT 比由低密度胶原构建的 CMT 的横截面积应力更低。我们还研究了电刺激对 CMT 内细胞排列和力产生的影响，并表明电刺激和附加负载的组合强烈改善了 CMT 的结构和功能。最后，我们证明了我们的技术适用于高通量监测 CMT 中自发性频率或收缩性的药物诱导变化，以及使用荧光染料进行高速钙动力学成像。总之，这些结果突出了该方法定量证明物理参数对心脏组织成熟度、结构和功能的影响的潜力，并为高通量、低体积筛选用于工程化心肌的研究开辟了可能性。
