Isolation and immortalization of human portal vein endothelial cells: A novel research tool for studying splanchnic vasculature.

Background & Aims: Cirrhosis profoundly impacts extrahepatic vasculature, particularly altering the portal venous system, leading to increased portal pressure, portosystemic collaterals, and portal vein thrombosis, which heightens morbidity and reduces survival in liver disease. Although intrahepatic vascular changes in cirrhosis are well studied, molecular insights into extrahepatic alterations in the splanchnic region remain limited owing to the inaccessibility of the human portal vein and suboptimal preclinical models. Here, we aim to isolate, characterize, and immortalize primary human portal vein endothelial cells (PVECs) to enhance understanding of pathophysiological changes during liver disease and establish a platform for future drug testing. Methods: PVECs (n = 12) and inferior cava vein (ICV, n = 9) endothelial cells (ECs) were isolated from human portal vein or ICV, obtained during hepatic transplantation, using trypsinization, mechanical scratching, and FACS. EC identity was confirmed through characterization of gene and protein marker expression as well as functional assays assessing angiogenic capacity (tube formation), migratory ability (wound closure), and acetylated low-density lipoprotein uptake. PVECs were immortalized (iPVECs) with lentiviral particles expressing the SV40 large T-antigen. Results: Isolated PVECs confirmed classical endothelial morphology and functionality, expressing hallmark proteins and functions. PVECs exhibited a distinct transcriptomic profile from ICVEC and systemic ECs, enriched in pathways for vascular remodeling and stress response. iPVECs retained endothelial identity and preserved the PVEC-specific transcriptomic traits across more than 20 passages. Conclusions: We successfully isolated, characterized, and immortalized PVECs, creating a novel tool to study splanchnic vascular diseases. These cells retain transcriptomic uniqueness distinct from systemic venous ECs, enabling investigation of vascular dysfunction mechanisms in liver disease and supporting translational research. Impact and implications: Portal hypertension and vascular complications are major drivers of morbidity in cirrhosis, yet extrahepatic vascular mechanisms remain poorly understood due to limited access to human portal vein tissue and inadequate models. By isolating, characterizing, and immortalizing primary human portal vein endothelial cells, we establish the first renewable, disease-relevant platform for studying splanchnic vascular biology. These immortalized portal vein endothelial cells preserve endothelial identity and transcriptomic signatures distinct from systemic venous cells, providing unique insights into vascular remodeling and stress responses in liver disease. This resource enables mechanistic discovery and drug testing aimed at improving outcomes in portal hypertension and related complications.