Vascular endothelial cells provide a critical interface between the vessel wall and blood borne elements. They modulate clot formation, vascular tone, chemotaxis, angiogenesis; and consequently, have the potential ability to aid in revascularization therapy, prosthetic cardiac valve or stent coverage, and as an autologous vector in gene therapy. In order to address these future goals, our study attempted to establish a reliable and reproducible system to culture endothelial cells from murine blood samples for use in further modeling studies.
Mouse blood was collected via intercardiac aspiration from CO2 anaesthetized animals. Anticoagulation, buffy coat suspension, cellular washing, plating and incubation were undertaken to optimize cell isolation. Additionally, cell specific marker panning with antibody coated magnetic beads were used in cell isolation. Various plating conditions, media growth factors, nutritional additives, and serum supplements were manipulated to optimize cell expansion. Phase Contrast microscopy, immuno-histochemical staining and flow cytometry were undertaken to confirm cellular characterization.
The murine blood outgrowth endothelial cells (BOECs) that we obtained take up acetylated low-density lipoprotein (AcLDL) and stained positive for multiple endothelial cell markers including von Willebrand factor (VWF), platelet/endothelial cell adhesion molecule (PECAM), vascular-endothelial (VE) - cadherin, and vascular cell adhesion molecule (VCAM). They are negative for CD11b - a marker of granulocytes, monocytes, and natural killer cells; and negative for CD14 - a marker of monocytes and polymorphonuclear leukocytes. Moreover, these cells demonstrate proliferative potential, tolerate cryopreservation, and are amenable to gene transfer with a retroviral vector for the expression of Green Fluorescent Protein (GFP) and human factor VIII.
A reliable and reproducible culture method was successfully developed to grow murine blood outgrowth endothelial cells. These cells have a potential use as a gene therapy delivery tool.
Murine BOECs can be utilized in modeling studies for gene therapy, limb and tissue revascularization, and coverage for prosthetic valves and stents.
A. Somani, None.