Biomechanical stimulation is a common strategy to improve the growth, maturation, and function of a variety of engineered tissues. However, identifying optimized biomechanical conditioning protocols is challenging, as cell responses to mechanical stimuli are modulated by other multifactorial microenvironmental cues, including soluble factors and biomaterial properties. Traditional bioreactors lack the throughput necessary for combinatorial testing of cell activity in mechanically stimulated engineered tissues. Microfabricated systems can improve experimental throughput, but often do not provide uniform mechanical loading, are challenging to use, lack robustness, and offer limited amounts of cells and tissue for analysis. To address the need for higher-throughput, combinatorial testing of cell activity in a tissue engineering context, we developed a hybrid approach, in which flexible polydimethylsiloxane microfabricated inserts were designed to simultaneously generate multiple tensile strains when stretched cyclically in a standard dynamic bioreactor. In the embodiment presented in this study, each insert contained an array of 35 dog bone-shaped wells in which cell-seeded microscale hydrogels can be polymerized, with up to eight inserts stretched simultaneously in the bioreactor. Uniformity of the applied strains, both along the length of a microtissue and across multiple microtissues at the same strain level, was confirmed experimentally. In proof-of-principle experiments, the combinatorial effects of dynamic strain, biomaterial stiffness, and transforming growth factor (TGF)-beta 1 stimulation on myofibroblast differentiation were tested, revealing both known and novel interaction effects and suggesting tissue engineering strategies to regulate myofibroblast activation. This platform is expected to have wide applicability in systematically probing combinations of mechanobiological tissue engineering parameters for desired effects on cell fate and tissue function. Impact Statement In this study, we introduce a dynamic bioreactor system incorporating microfabricated inserts to enable systematic probing of the effects of combinations of mechanobiological parameters on engineered tissues. This novel platform offers the ease of use, robustness, and well-defined mechanical strain stimuli inherent in traditional dynamic bioreactors, but significantly improves throughput (up to 280 microtissues can be tested simultaneously in the embodiment presented in this study). This platform has wide applicability to systematically probe combinations of dynamic mechanical strain, biomaterial properties, biochemical stimulation, and other parameters for desired effects on cell fate and engineered tissue development.

Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.

APPLIED BIOLOGICAL SCIENCES Correction for “Design and formulation of functional pluripotent stem cell-derived cardiac microtissues,” by Nimalan Thavandiran, Nicole Dubois, Alexander Mikryukov, Stéphane Massé, Bogdan Beca, Craig A. Simmons, Vikram S. Deshpande, J. Patrick McGarry, Christopher S. Chen, Kumaraswamy Nanthakumar, Gordon M. Keller, Milica Radisic, and Peter W. Zandstra, which appeared in issue 49, December 3, 2013, of Proc Natl Acad Sci USA (110:E4698–E4707; first published November 19, 2013; 10.1073/ pnas.1311120110). The authors note the following: “The reported conduction velocity of our human cardiac microwires, in Table 1, should read 25.1 ± 7.7 cm/s, rather than 47.4 ± 12.4 cm/s. This error was due to an unaccounted scaling factor during microscope imaging. We acknowledge Dr. Nenad Bursac for notifying us of this error. “The conduction velocity of Purkinje fibers, in Table 1, cited from a previous publication [Durrer D, et al. (1970) Total excitation of the isolated human heart. Circulation 41(6):899–912] should read ∼200 cm/s rather than ∼2000 cm/s. “The major conclusion of our manuscript, which is that we determine and use engineering design criteria for the formulation of cardiac microtissues from pluripotent stem cell-derived cardiomyocytes, is not impacted by this error.” The corrected table appears below.

Aortic valve homeostasis is mediated by valvular interstitial cells (VICs) found in spatially distinct and mechanically dynamic layers of the valve leaflet. Disease progression is associated with the pathological differentiation of VICs to myofibroblasts, but the mechanobiological response profiles of cells specific to different layers in the leaflet remains undefined. Conventional mechanically dynamic macroscale culture technologies require a large number of cells per set of environmental conditions. However, large scale expansion of primary VICs in vitro does not maintain in vivo phenotypes, and hence conventional macroscale techniques are not well-suited to systematically probe response of these cell types to combinatorially manipulated mechanobiological cues. To address this issue, we developed a microfabricated composite material screening array to determine the combined effects of dynamic substrate stretch, soluble cues and matrix proteins on small populations of primary cells. We applied this system to study VICs isolated from distinct layers of the valve leaflet and determined that (1) mechanical stability and cellular adhesion to the engineered composite materials were significantly improved as compared to conventional stretching technologies; (2) VICs demonstrate layer-specific mechanobiological profiles; and (3) mechanical stimulation, matrix proteins and soluble cues produce integrated and distinct responses in layer-specific VIC populations. Strikingly, myofibroblast differentiation was most significantly influenced by cell origin, despite the presence of potent mechanobiological cues such as applied strain and TGF-β1. These results demonstrate that spatially-distinct VIC subpopulations respond differentially to microenvironmental cues, with implications for valve tissue engineering and pathobiology. The developed platform enables rapid identification of biological phenomena arising from systematically manipulating the cellular microenvironment, and may be of utility in screening mechanosensitive cell cultures with applications in drug screening, tissue engineering and fundamental cell biology.

This paper reports the design and use of a novel high-throughput mechanobioreactor system for multifactorial investigations of cell-seeded engineered microtissues. Combinatorial experiments were conducted to test the independent and combined effects of mechanical strain, matrix stiffness, and a biochemical factor on the expression of α-smooth muscle actin (α-SMA), a pathological marker, in porcine aortic valve interstitial cells (VICs). We found that mechanical tension, transforming growth factor-β1 (TGF-β1), and matrix stiffness, all significantly increased α-SMA levels independently (p < 0.001, p < 0.001, p = 0.05, respectively), as well as in a coupled manner.

Poly(dimethylsiloxane) (PDMS)-based microdevices have enabled rapid, high-throughput assessment of cellular response to precisely controlled microenvironmental stimuli, including chemical, matrix and mechanical factors. However, the use of PDMS as a culture substrate precludes long-term culture and may significantly impact cell response. Here we describe a method to integrate polyurethane (PU), a well-studied and clinically relevant biomaterial, into the PDMS multilayer microfabrication process, enabling the exploration of long-term cellular response on alternative substrates in microdevices. To demonstrate the utility of these hybrid microdevices for cell culture, we compared initial cell adhesion, cell spreading, and maintenance of protein patterns on PU and PDMS substrates. Initial cell adhesion and cell spreading after three days were comparable between collagen-coated PDMS and PU substrates (with or without collagen coating), but significantly lower on native PDMS substrates. However, for longer culture durations (≥6 days), cell spreading and protein adhesion on PU substrates was significantly better than that on PDMS substrates, and comparable to that on tissue culture-treated polystyrene. Thus, the use of a generic polyurethane substrate in microdevices enables longer-term cell culture than is possible with PDMS substrates. More generally, this technique can improve the impact and applicability of microdevice-based research by facilitating the use of alternate, relevant biomaterials while maintaining the advantages of using PDMS for microdevice fabrication.