Developing a Soft Micropatterned Substrate to Enhance Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC-CM immaturity by introducing a novel physiologically relevant micropatterned substrate for long-term culture and maturation. A novel microfabrication technique combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue, respectively. Platinum electrodes integrated into the cell culture chamber enabled pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca ²⁺ transient, sarcomere orientation, distribution, and nucleus aspect ratio are analyzed in a 2D hiPSC-CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC-CMs for three weeks on a micropatterned PDMS substrate (2.5-5 µm deep, 20 µm center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, nucleus aspect ratio, contractility, and cytosolic Ca ²⁺ transient. The study provides significant insights into substrate stiffness effects on hiPSC-CM contractility and Ca ²⁺ transient at immature and mature states. Maximum contractility and fastest Ca ²⁺ transient kinetics occur in mature hiPSC-CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. This new substrate offers a promising platform for disease modeling and therapeutic interventions.