Abstract
Cardiospheres (CSs) are widely used to boost the pro-reparative potential of adult cardiac cells, mediated through their unique secretome profile. The original CS generation method relies on self-assembly of cardiac explant-derived cells (EDCs) on poly-D-lysine (PDL)-coated plates, but yields inconsistently sized spheroids, restricting broader applications. To address this, we employed ultra-low attachment (ULA) U-well plates to promote uniform spheroid assembly. We systematically compared CSs generated from mouse EDCs using the standard method, based on PDL-coated plates, and the alternative approach, based on ULA U-well plates. Both methods produced viable CSs mimicking the cardiac microenvironment, including mesenchymal cells/fibroblasts, smooth muscle, endothelial, and progenitor cells. PDL-formed CSs were characterized by size heterogeneity, increased stiffness, and reduced endothelial cell content. Despite that, they demonstrated elevated secretion of angiogenesis-related factors and robust proangiogenic potential in vivo. In contrast, generation of mCSs on ULA U-well plates resulted in the formation of soft spheroids with uniform size, enhanced vascularization (CD31+ cells), and increased MCP-1 secretion. In summary, the alternative U-well-based approach enables the generation of uniform spheroids with high spontaneous vascularization, while traditionally formed CSs using PDL-coated plates maintain their superior proangiogenic potential.