A novel cardio-vascular media enable to assess the complexity of multicellular organoids

Abstract Human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) provide a powerful in-vitro model to study cardiac physiology and disease. However, most existing models face two major limitations: the immature phenotype of hiPSC-CMs that does not recapitulate adult myocardium, and the absence of an authentic multicellular environment that restricts modeling of complex acquired cardiac diseases. While biochemical, mechanical, and metabolic cues have improved CM maturation, these approaches primarily target unicellular cultures. This study aimed to develop and characterize a novel maturation medium, Cardio-Vascular Medium (CVM), in combination with an assisted-assembly strategy of multicellular organoids, to enhance structural, metabolic, and functional maturation of hiPSC-derived cardiac tissue. hiPSCs were differentiated into CMs, endothelial cells (ECs), cardiac fibroblasts (CFs), and smooth muscle cells (SMCs). Organoids containing 10,000 cells in a defined ratio were generated. Organoids and 2D CM cultures were exposed to CVM, and maturation was assessed using RT-PCR, Western blotting, immunostaining, Seahorse metabolic assay, high-throughput all-optical functional analysis with optogenetic pacing, and proteomics including STRING network and GO enrichment analysis. CVM significantly enhanced CM maturation markers, with increased TNNI3 expression (~5 fold) compares to controls. Sarcomere structure was markedly improved, with sarcomere length increasing from 1.58±0.09 mm to 1.67±0.07 mm (p