Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely microscopic topography (3D shape resembling that of adult CM) and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate pillars of the desired 3D shapes, which would be used to mould the designed microwell arrays into a hydrogel. Polyacrylamide was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without comprising its capacity to modulate the stiffness. In this manner, individual parameters can be finely tuned independently within the hydrogel: the dimension of 3D shaped microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in an absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell Peer reviewed version of the manuscript published in final form at Biofabrication (2020). membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2- MYL7 proteins; while displaying higher anisotropism in comparison to pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiological or pathological microenvironment to an isolated single hiPSC-CM in absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision this system to be easily applied to other tissues and cell types where the influence of stiffness and cellular shape plays also an important role in its physiology.

Keywords: microfabrication, cardiomyocyte, physical interaction, cell shape, calcium handling, human induced pluripotent stem cells