Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells.
OBJECTIVE
A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h. This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells 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-crosslinked collagen.


RESULTS
At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v). Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h. The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped 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-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.
Krause
P
P
Zentrum Chirurgie, Georg-August-Universität Göttingen, Germany.
Markus
P M
PM
Schwartz
P
P
Unthan-Fechner
K
K
Pestel
S
S
Fandrey
J
J
Probst
I
I
D000818:Animals; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004727:Endothelium / Q000166:cytology*; D008099:Liver / Q000166:cytology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free