In this study, we propose a new method for bioprinting 3D Spheroids to study complex congenital heart disease known as discrete subaortic stenosis (DSS). The bioprinter allows us to manipulate the extrusion pressure to change the size of the spheroids, and the alginate porosity increases in size over time. The Spheroids is composed of human umbilical vein endothelial cells (HUVECs), and we demonstrated that pressure and time during the bio-printing process can modulate the diameter of the spheroids. In addition, we used Pluronic to maintain the shape and position of the spheroids. Characterization of HUVECs in the Spheroids confirmed their uniform distribution, making them a suitable model for understanding interactions between macrophages, endocardial endothelial cells (EECs), and DSS fibroblasts. The multiple cells typing in 3D spheroids extend the horizon to study the functional interactions between cells in promoting fibrosis and DSS pathology. Compared to traditional 2D cell cultures, the 3D spheroids model provide more relevant physiological environments, making it valuable for drug testing and therapeutic applications.