Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Abstract Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light‐mediated crosslinking, integrated with an elastomeric substrate featuring strain‐responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell‐derived cardiomyocytes (hIPSC‐CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC‐CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin‐43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC‐CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material‐based approaches for addressing existing challenges in hIPSC‐CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.