Microcarrier culture of vascular endothelial cells on solid plastic beads

Abstract

The culture of vascular endothelial cells on solid plastic beads is described. A greater than 30-fold increase in cell numbers was achieved in stationary culture medium. The inclusion of fibroblast growth factor slightly improved the rate of growth from low densities. Addition of fresh beads to colonized beads resulted in colonization of the newly introduced microcarrier. In common with the behaviour of endothelium in conventional culture, the cells cultured on beads changed from a fusiform to a polygonal shape after reaching confluence. Cell proliferation was also observed by [ 3H]thymidine autoradiography of DNA. The fraction of radiolabelled nuclei declined at confluence on each bead, indicating density-inhibition of growth. By electron microscopy, the cells displayed the typical ultrastructural appearance of endothelium. Following transfer of colonized beads to a chromatography column with slow perfusion of the bead bed, cell viability was maintained over a 24 h period and proportional synthesis of prostaglandin I 2 upon stimulation by ionophore A23187 was demonstrated. This simple microcarrier technique allows the generation of large numbers of vascular endothelial cells for subcellular fractionation with economical use of space and medium. When set up as a perfused bead bed, it offers possibilities for the short-term collection of concentrated endothelial metabolites.