Visualization of live endothelial cells ex vivo and in vitro

Abstract

The present study describes quick and effective methods that allow visualization of the vascular endothelium in living networks within dissected pieces of human tissue or in primary cultures containing heterogeneous cell populations. Fresh human uterine and subcutaneous gluteal fat tissues were directly labelled using fluorescently conjugated Ulex Europaeus Agglutinin I (UEA-1) to visualize the three-dimensional nature of the vascular network. Using conventional epi-illuminescence microscopy, the convoluted architecture demonstrating branch points within capillaries, between capillaries and larger vessels, were clearly observed in uterine and subcutaneous gluteal fat samples. In adult endometrial tissue where angiogenesis occurs on a monthly basis, complex anastamosis of vessels and tenuous structures were clearly seen. Three-dimensional rendered surface models formed by examination of confocal z-stacks demonstrated the existence of the lumen within microvessels. Tissue prelabelled with UEA-1 was used to assist and verify the presence of endothelial cells in culture, during and after the isolation procedure. Additionally, UEA-1 was added to a uterine fibroblast-microvascular-endothelial cell coculture model to allow daily vital observations of changes in the phenotype of the endothelium. The simple techniques described here demonstrate the ease with which fluorescently labelled UEA-1 can be used as a vital marker of endothelial cells either in tissue or in a tube-forming human uterine microvascular culture model.