Isolation and Characterization of Porcine Endocardial Endothelial Cells

The heart contains six different types of endothelial cells, each with a unique function. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of this cell line, we developed a protocol for isolating EECs from porcine hearts and detailed our methodology for establishing populations of EECs through cell sorting. Additionally, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs were slightly smaller than HUVECs, and they stained positively for classic endothelial phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs, yet migrated more slowly to cover a scratch wound assay. Finally, the EECs maintained their robust endothelial phenotype (expression of CD31) through more than a dozen passages. In contrast, the HUVECs showed significantly reduced CD31 expression in later passages. These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell lines when studying or modeling a disease of interest. Impact statement Many researchers model cardiovascular disease via tissue engineering to determine disease etiology on the cellular and molecular level. However, researchers usually rely on commercially-available cell lines, which can result in utilizing cells that are not specific to the region of the heart being modeled and could lead to incorrect conclusions. By providing a detailed protocol for isolating and purifying endocardial endothelial cells, we are enabling other researchers to access this cell line and model cardiovascular disease states more accurately. ### Competing Interest Statement The authors have declared no competing interest.