Hepatocyte primary culture bioassay: a simplified tool to assess the initiation of the liver regeneration cascade.
The objective was to develop and optimize a hepatocyte primary culture bioassay to detect proliferative factors (PF) in the serum or plasma of partially hepatectomized (PHX) rats to serve as a tool to assess the initiation of the liver regeneration cascade. The bioassay detects PF by measuring hepatocyte proliferation through directly counting increases in viable cell number over the culture period using a hemocytometer. Hepatocytes were obtained using a two-step collagenase perfusion procedure. The purified hepatocytes (>80% viability, >95% parenchymal cells) were seeded into 6-well culture plates and allowed to attach overnight. The unattached cells were washed out, and the starting cell count was determined from three randomly selected wells after trypsin digestion. Sera from 2/3 PHX rats at 1-6 h postPHX was added to the culture. With a medium change at 24 h, the final cell counting was performed at 48 h. The net cell proliferation was expressed as the difference between the counts at 48 h and starting h. The optimized assay conditions could detect an increase of PF in PHX rat serum between 1 and 4 h after PHX (peaking at 4 h). The bioassay showed both a qualitative and quantitative sensitivity to distinguish between the PF levels in 1/3 and 2/3 PHX rats.
Wang
H H
HH
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Lautt
W W
WW
D000818:Animals; D002452:Cell Count; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D006498:Hepatectomy; D008099:Liver / Q000502:physiology*; D008115:Liver Regeneration; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley