3D droplet-based microphysiological niches drive early maturation transcriptional remodeling in human iPSC-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for cardiac disease modeling and drug screening but remain limited by immature phenotypes and the inability of conventional culture systems to precisely control extracellular matrix (ECM) cues at single-cell resolution. Here, we present a Matrigel-containing microfluidic droplet platform for single-cell hiPSC-CM culture. Droplets were produced by flow-focusing, enabling single-cell encapsulation, controlled matrix exposure, live-cell imaging, and downstream molecular analysis. Systematic optimization identified 10% and 30% Matrigel dilutions as the optimal ECM concentration for bulk hiPSC-CMs culture in 2D plate format promoting hiPSC-CMs spreading and elongation. Encapsulation of 30% Matrigel in droplets maintained >80% hiPSC-CMs viability during 24-h on-chip culture. Using bulk RNA sequencing of cells pooled from droplets we show that 24-h 3D droplet culture induced a distinct transcriptional state compared with conventional 2D culture and freshly encapsulated 3D droplet control. Differential gene and pathway enrichment analyses revealed activation of mitochondrial oxidative phosphorylation and ATP biosynthesis programs, alongside activation of early tissue-like cardiovascular signaling and remodeling pathways. These findings indicate that controlled ECM presentation in uniform 3D droplet microenvironment can initiate early metabolic maturation-associated transcriptome remodeling in hiPSC-CMs. Collectively, this work establishes a droplet-based platform for hiPSC-CM culture providing a foundation for next-generation microphysiological systems used in cardiac disease modeling, cardiotoxicity screening, and mechanistic studies of cardiomyocyte maturation.