Serial subculture and relative transport of human endothelial cells in serum-free, defined conditions

Abstract

Endothelial cells bind, process, and transport bioactive molecules and thus provide an interactive interface between the plasma and adjacent tissues. Various hormones and factors induce endothelial cells to synthesize and secrete interactive factors. However, study of endothelial cell synthesis, processing, and transport of these bioactive molecules has been impeded because of the serum requirement for cell growth. Many of these bioactive molecules are derived from or are modified by serum components. We have developed a short-term culture system that supports sequential subculturing of endothelial cells in a serum-free culture medium on a defined extracellular matrix. The cells have a doubling rate of 33 h and the total cell number can be expanded more than 800-fold. Expression of specific markers; factor VIII related antigen-von Willebrand factor, Wiebel-Palade bodies, a cobblestone appearance of confluent cell monolayers, and angiotensin-converting enzyme activity confirm the normal morphologic integrity and biochemical function of these cultures. Using this defined serum-free medium, we have grown human endothelial cell monolayers on porous polycarbonate membrane inserts, thereby creating an upper and a lower chamber that models the vascular architecture and demonstrates an inverse relationship between transport and molecular weight. By eliminating serum components, this model system should facilitate the study of endothelial cell binding, metabolism, and transport of bioactive molecules and may contribute to a better understanding of the blood-tissue interface.