Background Primary porcine aortic endothelial cells (PAECs) are currently the main target cells for studying human-to-pig xenoreactive immune responses, particularly for measuring the levels of preformed natural antibodies (NAbs). However, primary PAECs have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (iPAECs) to facilitate in vitro studies. Methods Isolated and cultured primary aortic endothelial cells from different gene-edited pigs (WT, GTKO, TKO: GTKO/b4GalNT2KO/CMAHKO): Primary porcine aortic endothelial cells were transfected with lentiviral containing the SV40 large T antigen (SV40LT) and selected on the basis of morphological and phenotypical features. Expression of Gal, Sda, and Neu5Gc on iPAECs from different types of knockout pigs were analyzed using flow cytometry. Anti-pig IgM/IgG binding and complement- dependent cytotoxicity (CDC) to these iPAECs was measured by flow cytometry using pooled rhesus monkey sera (n=20) or human sera (n=20). Results In terms of cell morphology, compared to primary cells, iPAECs were rounder in shape, larger in individual size, more transparent, and had indistinct cell boundaries. iPAEC cell lines could be expanded for up to 20 passages without signs of senescence and with preserved characteristics of primary cells, such as cobblestone morphology and staining for CD31 (the purity of CD31 staining is greater than 95%). The expression of αgal, Sda and Neu5Gc antigens in iPAECs is not significantly different from that in primary aortic endothelial cells. In addition, both the antibody binding (IgM and IgG) and CDC of rhesus monkey and human against WT pig iPAECs were very high (GMean of IgM: ~8000 and ~9000; GMean of IgG: ~200 and ~600; CDC: >90%). In the case of both humans and rhesus monkeys, the binding of serum IgM and IgG to GTKO pig iPAECs (Gal deletion) was significantly lower than that to wild-type pig iPAECs (P<0.01). The antibody binding (IgM and IgG) and CDC of human serum against TKO pig iPAECs was markedly decreased after the deletion of Gal, Sda, and Neu5Gc in pigs. Unlike the results we obtained for pooled human serum, the binding of rhesus monkey serum IgM/IgG antibody to TKO pig iPAECs was significantly lower than that to pig iPAECs from WT, GTKO pigs.; Hhowever, the cytotoxicity of monkey serum against TKO pig iPAECs was only slightly lower than that to GTKO pig iPAECs (P<0.05). All the above results were similar to our findings using PBMCs or primary PAEC from the same gene-edited pigs. Conclusion In conclusion, iPAECs cells are a newly established porcine aortic endothelial cell lines morphologically, phenotypically and functionally similar to primary cells. These cell lines should therefore provide valuable tools for in vitro studies of the interactions between the human immune system and the porcine endothelium.