Enhanced endothelial cell adherence and spreading on basement membrane matrix coated polystyrene thin films suitable for LM, LV-HR-SEM, and HVEM imaging

Abstract

Carefully controlled conditions of culture are essential to the in vitro establishment of human primary endothelia] cell cultures. In order to facilitate rapid adherence, spreading, and the development of an in vivo like polygonal pattern of growth, the selection of an appropriate substrate is particularly important. Typically polystyrene “tissue culture” grade dishes subsequently coated with basement membrane proteins, BMM, (collagen, fibronectin, and laminin), or other combinations of extracellular matrix proteins provide an adequate substrate. Correlative light microscopic and electron microscopic examination of cultured whole cell preparations requires that for TEM, HVEM, and STEM, the ceils be grown on a thin film. In addition to the usual requirements of strength and stability, as well as compatability with fixation, staining and dehydration procedures, the films need to be as optically strain free as possible to permit video-enhanced interference or phase based LM.In preliminary studies we found traditional EM substrates such as formvar and collodion, even when coated with various combinations of matrix proteins, not to be ideal substrates (Fig. 1). Endothelial cell adhesion was reduced, spreading was incomplete even after 4‒6 hours, the rate of cell division was slowed, and the time to confluence was prolonged.