RESEARCH SUMMARY
This study investigated whether controlled hydrostatic pressure could promote structural, functional, and metabolic maturation of engineered cardiac tissues derived from human pluripotent stem cells. ECTs containing hiPSC-derived cardiomyocytes, endothelial cells, mural cells, collagen I, and Matrigel were first cultured for two or three weeks and then subjected to different pressure-training regimens. Continuous cyclic pressure at physiological or supraphysiological levels did not improve cardiomyocyte content, whereas intermittent stimulation revealed a strong dependence on pressure magnitude, duration, and developmental timing. The optimized protocol consisted of 50 kPa hydrostatic pressure for 1 hour per day over 3 days after 2 weeks of pre-maturation. This treatment increased cardiomyocyte density and alignment, collagen organization, expression of mature cardiac markers including TNNT2, TNNI3, and CACNA1C, and produced a positive force-frequency relationship indicative of improved contractile maturation. Pressure-trained ECTs also exhibited enhanced calcium handling, including greater calcium-transient amplitude and faster calcium uptake and release, together with increased SERCA2 and PLN expression. Metabolic maturation was demonstrated by increased mitochondrial content, larger and less circular cardiomyocytes, increased maximal oxygen consumption, and greater ATP production. Endothelial cells were essential for these effects: ECTs lacking endothelial cells failed to develop the structural, calcium-handling, and force-frequency improvements induced by pressure, while single-cell RNA sequencing showed endothelial-cell-dependent enhancement of cardiac contraction, calcium signaling, structural organization, and metabolic pathways. Combining pressure training with an additional week of dynamic culture produced a distinct vascular network and extended positive force-frequency behavior to approximately 2 Hz. Transplantation of pressure-trained ECTs into a rat myocardial infarction model improved left ventricular ejection fraction, fractional shortening, and ventricular remodeling, and human myocardial tissue was detected at the graft site. Patient-derived familial dilated cardiomyopathy ECTs showed a much weaker maturation response to the same pressure regimen, suggesting potential utility for disease modeling. Overall, the study demonstrates that brief, high-intensity intermittent hydrostatic pressure can promote endothelial-cell-dependent maturation of engineered human cardiac tissue and improve its regenerative potential.
A CellScale MechanoCulture TR was used to apply controlled hydrostatic pressure training to human iPSC-derived engineered cardiac tissues. ECTs were generated in custom PDMS molds from approximately six million differentiated cardiovascular cells combined with collagen I and Matrigel and cultured for 14 or 21 days before stimulation. The tissue-containing molds were transferred into the MechanoCulture TR chamber, fully surrounded with culture medium, and the system was operated inside a tissue-culture incubator. The MCTR applied cyclic hydrostatic pressure by changing the pressure inside the sealed culture chamber without directly stretching or platen-compressing the ECTs. Each pressure cycle consisted of a 0.2-second compression phase, 0.1-second hold, 0.2-second recovery, and 0.5-second rest, corresponding to a 1-second cycle. The researchers initially compared continuous cyclic loading at 10 and 50 kPa and then evaluated intermittent pressure magnitudes of 10, 50, and 100 kPa. Intermittent loading at 50 kPa produced the strongest maturation response. Duration experiments showed that 1 hour per day for 3 days after 2 weeks of pre-maturation was superior to 8 days of stimulation, as prolonged pressure increased apoptosis and reduced tissue quality. The optimized MCTR protocol therefore used 50 kPa for 1 hour per day on three consecutive days after two weeks of ECT culture. MechanoCulture TR pressure training increased cardiomyocyte alignment and density, improved calcium handling and force-frequency behavior, increased mitochondrial and metabolic maturation, and altered maturation-associated gene expression. Experiments using ECTs without endothelial cells showed that these benefits depended strongly on endothelial-cell-mediated mechanotransduction. Pressure-trained ECTs were subsequently used in vascularization, disease-modeling, and rat myocardial-infarction transplantation experiments, establishing the MCTR as the core mechanical-conditioning platform for producing more mature engineered cardiac tissue.
