Development of a novel <i>In Vitro</i> Co-culture system for discrete subaortic stenosis

Discrete subaortic stenosis (DSS) is a severe congenital heart condition that results in the formation of a fibrous membrane in the left ventricular outflow track (LVOT). While DSS is surgically treated, it is frequently associated with a high rate of recurrence, necessitating multiple surgeries. The surgical burden of DSS can be reduced by implementing targeted drug therapies based on the underlying molecular mechanisms. There are multiple cell types within the LVOT, consisting of fibroblasts and endocardial endothelial cells (EECs), organized in a complex 3D space. Our objective of this study was to develop a 3D system for the concurrent coculture of fibroblasts and EECs, as a first step in the development of a tool to better understand the cellular communication between these two cell types. To accomplish this objective, we used extrusion-based bioprinting to fabricate 3D discs. Extrusion-based bioprinting was used to generate a 3D disc with fibroblasts and EECs distributed in different configurations within the 3D disc. We demonstrated that the fibroblasts and the EECs maintained viability as a function of time for up to 4 days under static (this is extra spacing here) conditions. Furthermore, to simulate the wall shear stress conditions in the LOVT, a cone and plate bioreactor was used in conjunction with the 3D bioprinted disc for a culture period of up to 24 hours. We demonstrated that EECs maintained CD31 expression for up to 24 hours when cultured within the 3D discs under conditions of elevated shear stress. Collectively, our results demonstrate the initial success of the 3D bioprinted disc model as a potential tool for studying DSS. While additional optimization and validation studies are required, the model described in this study has the potential to provide insight into the underlying molecular mechanism of DSS disease phenotype and lead to the development of targeted therapies for the treatment of this challenging congenital heart condition.