Introduction:
Engineered Heart Tissue (EHT) derived from human induced pluripotent stem cells (hiPSCs) holds significant potential for repairing congenital heart defects. Our research aims to develop a pulsatile Fontan tunnel to improve outcomes in patients with Fontan circulation. While dynamic culture conditions have been shown to enhance tissue maturation, particularly in strip-like EHTs, their impact on tubular constructs remains less explored. Building on prior findings, we compared static and dynamic culture conditions for tubular EHTs (tEHTs), focusing on pressure generation, structural properties, and electrophysiological function.
Methods:
Thirteen tEHTs, each casted from 20 million hiPSC-derived cardiomyocytes, were cultured for 30 days in a custom-engineered bioreactor. Seven tubes were maintained under dynamic conditions with mechanical stimulation on a horizontal shaker (0.5 Hz, 15°) and six under static conditions. Intratubular pressure was measured several times during the culture period (dynamic max. pressure 0,6mmHg, static max. pressure 0,3mmHg). At the endpoint, histological analyses were performed to assess structural organization and extracellular matrix (ECM) composition. Additionally, two dynamically cultured and two statically cultured tEHTs were tested in an organ bath system for pharmacological stimulation,including force measurements (mN), and action potentials were measured using sharp microelectrode recordings to evaluate electrophysiological function.
Results:
Dynamically cultured tEHTs exhibited superior mechanical and structural properties compared to their static counterparts. Pressure measurements during the 30-day culture period demonstrated consistent improvements in pressure resistance in the dynamic group, indicating enhanced mechanical integrity. Histological analyses revealed increased cardiomyocyte alignment and greater ECM organization in dynamically cultured tubes.
Organ bath experiments showed increased contractile force generation in dynamically cultured tEHTs compared to static controls, reflecting improved functional maturation. Electrophysiological measurements using sharp microelectrode showed stable, synchronous action potentials.
Conclusion:
Dynamic culture conditions enhance the mechanical strength and functional performance of tEHTs, confirming their previously observed benefits. These findings underscore the impact of dynamic stimulation in promoting tissue maturation and structural stability. Future research will investigate additional strategies, such as pulsatile mechanical conditioning and metabolic modulation, and explore combinations of these approaches to achieve optimal outcomes. These efforts aim to refine further tEHT development for clinical applications, particularly in creating pulsatile Fontan tunnels for patients with univentricular hearts.
