Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells

Background/Aims: A major problem in rat liver endothelial cell culture is t he rapid loss of cells after 48 h, This study aimed to develop a protocol t hat allowed easy maintenance and proliferation of sinusoidal endothelial ce lls in serum-free culture for 5-6 days. Methods: Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslin ked collagen, Results: At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth facto r; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes prov ed to be the most effective growth stimulus; it increased thymidine incorpo ration, DNA content and cell number per dish with a half-maximal effect at 20% (v/v), Cell proliferation was also observed with either vascular endoth elial growth factor, phorbol ester or conditioned media from FAO or HEPG2 l iver cell lines provided the cultures were additionally supplemented with 1 % newborn calf serum. Vascular endothelial growth factor was detected in al l conditioned media. In the absence of hepatocyte-conditioned medium, 1% se rum helped to maintain cultures; it itself exerted a low proliferative effe ct. Higher serum concentrations (>5%), however, led to cell loss after 48 h , The numerous sieve plates of 6-h-old cells progressively disappeared duri ng culture and were replaced by randomly distributed pores, which later gro uped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein, Conclusions: The study shows that cultured hepatocytes secrete growth-promo ting substances that stimulate in vitro endothelial cell proliferation in t he absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulat ions should facilitate future research on liver endothelial cells in mono- or coculture.