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Prevention of changes in Endothelial Cells Cultured in High Glucose

The vascular endothelium is the interface with hyperglycemia in uncontrolled diabetes mellitus and its complications, including nephropathy, myocardial infarction, blindness, and gangrene. To mimic the vascular endothelium in a controlled experimental situation, we use the endothelial cell line (CRL 1998) obtained from The American Type Culture Collection. Cells are grown to confluency in Media 199 containing 10% fetal calf serum and gentamicin (50mg/ml). The growth media is replaced with experimental media containing high glucose (30mM), insulin (20 Units/ml), or heparin (500 Units/ml) either separately or in combinations. Cells are briefly trypsinized, pelleted, embedded, and sectioned with a Diatome diamond knife for TEM or for SEM, grown on 0.45um filters, followed by fixation and air-drying in situ: this approach to culture allows cells to obtain a polar, more natural phenotype than monolayers on glass or plastic. Incubation in high glucose results in loss of confluency, production of intercellular gaps, and fewer mitotic figures.