Model for assessment of endothelial cell function and viability using the MTT dye test and [3H

Abstract

To establish a reproducible in vitro model for evaluating endothelial cell function in clinical disease states. A prospective study of human umbilical vein endothelial cells (HUVEC) isolated and cultured. Department of Haematology, University of Yaounde I, Cameroon and Department of Medicine University of Newcastle-upon-Tyne, England. The optimum initial cell seeding concentration, for maximal conversion of the formazan dye was 40 x 10(3) cells/well, after 24 hours incubation. At concentrations above 40 x 103 cells/well some inhibition of dye conversion occurred. The conversion of formazan dye was directly proportional to cell numbers for the first 48 hours only, at all cell concentrations. Thereafter, cell metabolism appeared to be inhibited. Third passage endothelial cells (ECs) were exposed to a range of lipopolysaccharide (LPS) concentrations for one and 24 hours, prior to performing the MTT dye test. Dye conversion was observed after one hour at even the lowest concentration of LPS (0.1 microgram/ml), to 49.9% +/- 5.6% of unperturbed control EC, with 10 x 10(3) initial seeding numbers. After 24 hours perturbation a small but statistically significant further inhibition was observed. [3H] thymidine incorporation studies indicated that the lowest LPS concentration tested (0.1 microgram/ml) had a stimulatory effect on DNA synthesis at the higher cell concentration (20 x 10(3) cells/well). In the range of 1 to 100 micrograms/ml of the LPS tested, there was increased DNA synthesis at all cell numbers. The model may be used to monitor the effects of other agents which are known to, or could be associated with, alterations in endothelial cell function and will serve in mimicking clinical situations including hyper coagulable states.