A method to reduce glutathione peroxidase levels in primary endothelial cell cultures

The purpose of this study was to develop culture conditions that would reduce glutathione peroxidase activity in bovine mammary endothelial cells. Conditions of reduce glutathione peroxidase activity were produced in vitro by culturing cells in selenium-deficient media. Low selenium levels were achieved by reducing serum concentrations; however, levels of essential growth factors also were reduced by this method. Therefore, cell proliferation was promoted by supplementation with combinations of defined serum components including insulin, transferrin, linoleic acid, bovine brain extract, and human epidermal growth factor. Out of seven different formulas tested, F12K medium containing 2% fetal bovine serum, insulin, transferrin, and linoleic acid was found to be conducive for cell proliferation. Upon confluence, endothelial cells cultured under these conditions consistently displayed short passage rates, consistent cell numbers, and classic `cobblestone' morphology when grown in the presence or absence of supplemental selenium. Additionally, these cells retained typical endothelial cell characteristics such as uptake of 1,1 theta -dioctadecyl-3,3,3 theta ,3 theta -tetramethylindo-carbocyanine perchlorate acetylated low-density lipoprotein and the expression of cell adhesion molecules and von Willebrand Factor.