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Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising platform for cardiac tissue engineering, disease modeling, and drug discovery. However, their limited structural and functional maturity remains a significant barrier to clinical and research applications. This thesis addresses this challenge by integrating mechanical property characterization, microfabrication of biomimetic substrates, and quantitative assessment of mechanical output to enhance the maturation of hiPSC-CMs. The study first explores the mechanical properties of myofibrils extracted from hiPSC-CMs using atomic force microscopy (AFM) and the quasi-linear viscoelastic (QLV) model. Results indicate that the viscous response of these myofibrils is independent of strain levels, particularly between 40% to 70%, validating the use of the QLV model to predict mechanical behavior. The myofibrils showed an elastic modulus of 9.78 ± 5.80 kPa, consistent with values reported for adult human cardiac tissue. Additionally, comparisons revealed that hiPSC-CM myofibrils exhibit slower relaxation than those extracted from porcine tissue, highlighting distinct mechanical characteristics important for therapeutic development. Further, the thesis introduces a micropatterned substrate with tunable stiffness integrated with fluorescent nanobeads for precise traction force microscopy (TFM). Optimal substrate dimensions were identified—20 micrometers center-to-center spacing and 2.5 to 5 micrometers depth, with 2 to 5 kPa stiffness—significantly enhancing hiPSC-CM alignment, elongation, mitochondrial density, and contractility. Long-term culture experiments demonstrated that cells cultured for three weeks achieved optimal maturation, indicated by increased sarcomere length and improved contractile parameters. An advanced workflow combining TFM, dual-plane imaging, and finite element analysis (FEA) quantified hiPSC-CM contractile behavior, highlighting the critical roles of substrate stiffness and micropatterning in modulating cellular mechanics. Micropatterning improved directional force generation, while stiffer substrates modeled pathological conditions, promoting increased stress generation and altered metabolism. Collectively, this integrated approach significantly advances the understanding and engineering of hiPSC-CM maturation, offering powerful tools for cardiac tissue engineering, disease modeling, and therapeutic development.