Bioengineering the Extracellular Matrix to Improve the Cellular and Molecular Physiology of Cells In Vitro

Stem cells hold great promise for improving the predictive power of preclinical in vitro drug screens and the translation of new technologies from bench to the bedside. However, traditional in vitro cultureware often fails to recapitulate the complex organization and interplay of cells and the extracellular matrix (ECM), failing to mimic critical in vivo phenotypes. Here, we present a novel biomimetic substrate with submicron topographies that is capable of mimicking the mechanical and structural cues of the ECM while benefiting from high-precision, easy-to-reproduce, and scalable photolithography-based fabrication techniques. We incorporate the substrate into SLAS/ANSI-format microplates for use in standard industry endpoint assays and high-throughput automated platforms. Additionally, this substrate is capable of providing an active mechanical loading environment to the monolayer of cells for more physiological in vitro conditioning. Here, we focus on how structural alignment can enhance development at the cellular level and improve the expression of different genes at the molecular level for improved maturity of human induced pluripotent stem cells (hiPSCs). We demonstrate that hiPSC-derived cardiomyocytes (hiPSC-CMs) grown on this biomimetic surface exhibits in vivo-like myofibril alignment, sarcomere spacing and width, and expression of CM-specific protein isoforms that are present only in mature myocytes. Similarly, we demonstrate that hiPSC-derived neurons show enhanced neurofilament alignment, faster migration and neurite outgrowth speed, as well as improved cell-cell communication and signaling. We evaluate the electrophysiological response of these cells through patch clamp and MEA platforms demonstrating more adult-like action potentials. Lastly, we confirm the utility of our approach by showing phenotype enhancement in other adherent mammalian cell types. We conclude that our approach is a reliable and scalable method for re-creating specific aspects of the ECM to enhance the development and maturation of stem cells.