Effects of Physiologic Mechanical Stimulation on EmbryonicChick Cardiomyocytes Using a Microfluidic Cardiac Cell Culture Model

摘要

Hemodynamic mechanical cues play a critical role in the early development and functional maturation of cardiomyocytes (CM). Therefore, tissue engineering approaches that incorporate immature CM into functional cardiac tissues capable of recovering or replacing damaged cardiac muscle require physiologically relevant environments to provide the appropriate mechanical cues. The goal of this work is to better understand the subcellular responses of immature cardiomyocytes using an in vitro cardiac cell culture model that realistically mimics in vivo mechanical conditions, including cyclical fluid flows, chamber pressures, and tissue strains that could be experienced by implanted cardiac tissues. Cardiomyocytes were cultured in a novel microfluidic cardiac cell culture model (CCCM) to achieve accurate replication of the mechanical cues experienced by ventricular CM. Day 10 chick embryonic ventricular CM (3.5 × 10⁴ cell clusters per cell chamber) were cultured for 4 days in the CCCM under cyclic mechanical stimulation (10 mmHg, 8–15% stretch, 2 Hz frequency) and ventricular cells from the same embryo were cultured in a static condition for 4 days as controls. Additionally, ventricularcell suspensions and ventricular tissue from day 16 chick embryo werecollected and analyzed for comparison with CCCM cultured CM. The geneexpressions and protein synthesis of calcium handling proteins decreasedsignificantly during the isolation process. Mechanical stimulationof the cultured CM using the CCCM resulted in an augmentation of geneexpression and protein synthesis of calcium handling proteins comparedto the 2D constructs cultured in the static conditions. Further, theCCCM conditioned 2D constructs have a higher beat rate and contractilityresponse to isoproterenol. These results demonstrate that early mechanicalstimulation of embryonic cardiac tissue is necessary for tissue proliferationand for protein synthesis of the calcium handling constituents requiredfor tissue contractility. Thus, physiologic mechanical conditioningmay be essential for generating functional cardiac patches for replacementof injured cardiac tissue.