Development of Pyramidal Microwells for Enhanced Cell Spheroid Formation in a Cell-on-Chip Microfluidic System for Cardiac Differentiation of Mouse Embryonic Stem Cells.

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine.