Establishment of the permanent microvascular endothelial cell line PBMEC/C1-2 from porcine brains

Porcine brain microvascular endothelial cells (PBMEC) were isolated from fresh brains by several enzymatic digestion steps followed by a gradient centrifugation. Cells of the primary culture were transfected with pRNS-1, encoding for the small and large T-antigens of SV 40, by means of lipofection. After selection with G-418, clones were isolated and one clone, PBMEC/C1-2, was further characterized by microscopic examination of morphology, immunofluorescence, and lectin binding. PBMEC/C1-2 was cultivated for nearly 1 year with 250 cumulative population doublings and showed the typical morphology of capillary endothelial cells in vitro. Postconfluent cultures showed the formation of a tubular network as a second layer, which is characteristic of capillary endothelial cells. SV 40 T-antigens were present in the nuclei and the cells showed the typical granular staining of von Willebrand factor (vWF) and were positive for Bandeiraea simplicifolia isolectin B4. Furthermore, PBMEC/C1-2 exhibited the uptake of acetylated LDL, expression of nonmuscular- and smooth-muscle-type myosins, and the presence of the blood-brain barrier-associated markers gamma-glutamyltranspeptidase (gamma-GT), the glucose transporter Glut-1, and apolipoprotein A-1. In addition, enzymatic activity of gamma-GT and alkaline phosphatase could be detected in cell suspensions. In summary, PBMEC/C1-2 represents an immortalized PBMEC line exhibiting endothelial characteristics as well as typical markers of the blood-brain barrier.