Establishment of Techniques for Primary Isolation, Characterization, and Long-Term Maintenance of Porcine Endothelial Cells

Abstract

The long-term maintenance of highly purified primary porcine endothelial cells(pEC) is eventually required for xenotransplantation and stem cell research. However, primary isolated cells, after four to five passages, are commonly used even without correct characterization. Therefore, along-term maintenance system of pEC was established. This system produces highly-purified and well-characterized pEC and functions property in order tofacilitate the studies of xeno-immune responses. Porcine thoracic aorta was dissected free of connective and adipose tissues, and aortic endothelium was digested and scraped mechanically. Isolated cells were filtered with nylon mesh, seeded on culture dishes, and maintained with specialized culture medium. Their function and characteristics were analyzed during long-term culture by immunohistochemistry, FACS analysis, uptake ability of acetylated low density lipoproteins(LDL), and cell proliferation assay. For over nineteen passages, cells showed a typical cobble-stone shape of endothelial cells and were endowed with the functional properties of endothelial cells, which could uptake acetylated low density lipoproteins(LDL). They also showed characteristic properties of endothelial cells, which could express endothelial specific markers, von Willebrand factor(vWF), and CD31 (PECAM). Using flow cytometry, it was shown that over 95% of cells were positive in CD31 and over 90% in Dil-ac-LDL for over fifteen passages. Consequently, the cells showed high purity and functioned as porcine endothelial cells even with long-term maintenance. A simplified and reliable primary isolation, long-term maintenance, and characterization system of porcine primary endothelial cells is provided. This system facilitates the study of the role of the endothelial response in human-porcine xenotransplantation and stem cell research.