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.

A chemo-mechanical free-energy-based approach to model durotaxis and extracellular stiffness-dependent contraction and polarization of cells

“…Cytoskeletal tension is fundamental to many cellular processes such as cell motility [1], cytokinesis [2], tissue morphogenesis [3][4][5][6] and remodelling, as well as pathological processes such as tumour growth, metastasis [7] and fibrosis [8]. A number of cells such as fibroblasts, cardiomyocytes, epithelial cells, endothelial cells and smooth muscle cells develop significant contractile forces as part of their physiological function [9][10][11][12].…”

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue

“…Finally, constrained gel compaction is the primary effector of cell and fiber alignment in threedimensional engineered tissues, and comparisons between aligned and isotropic ECTs show that alignment leads to increased twitch force. 16 All these factors associated with differential gel thickness can and will play into the contractile force measured for gel-based ECTs by any method [16][17][18][19][20][21][22] and need to be considered when interpreting the data.…”

“…Tissue-level assays have also been developed and include free-floating tissue samples, as well as anchored tissues of both macro-and microscales that have proven useful for investigating the role of alignment, 16 matrix stiffness, 17 and matrix composition. 18 However, free-floating assays do not measure cell forces directly, instead rely on theory to relate the tissue compaction to cell forces.…”

“…19,20 In addition, while macrosized tissues 16 are easy to prepare and conducive to force measurement without a highly specialized equipment, they are low throughput, and screening many factors quickly becomes cumbersome and taxing on resources. Conversely, microsized tissues enable high-throughput screening but generally require microfabrication 17,21 or the use of specialized equipment. 22 Herein, we propose and validate a new approach, which we term tissue contraction force microscopy, inspired by cell traction force microscopy that combines simplicity of sample preparation and the use of common laboratory equipment.…”

Engineering Cardiovascular Regeneration

“…Various systems have been developed to fix the remodeling tissue and provide the mechanical strain necessary for cell orientation between the fixation points. The setup ranges from Velcro-coated glass tubes [94], rings [95], Flexcell chambers [97], and mesoscopic micropost arrays [98] to newer systems in which the fixation posts serve as means to measure contractile force [99,100] (overview in [48]). For cardiac repair, the hydrogel systems can be upscaled, either by employing larger casting molds [101] or assembling of several smaller elements [102].…”