A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).