Abstract: Objective To establish the protocol for the separation of hepatocyte (PC), hepatic stellate cells
(HSC), sinusoidal endothelial cells (SEC), and Kupffer cells (KC) from liver of rats. Methods Liver single-
cells suspension was obtained by EGTA/collagenase perfusion. After low speed centrifugal separation of PC,
the nonparenchymal cells (NPCs) portion was added with pronase E to disrupt the PC. Subsequently, HSC,
SEC and KC were purified by two steps of density gradient centrifugation using Nycodenz and Percoll plus
selective attachment, and the cells phenotype and function of changes after cuture were then observed. Results
The average yield of isolated PC, HSC, SEC, and KC were (26 ± 9.2) × 10 6 , (1.5 ± 0.2) × 10 6 , (7.4 ± 1.5) ×
10 6 and (3.15 ± 0.9) × 10 6 cells per gram of liver tissue, respectively. The average viability of isolated PC, HSC,
SEC, and KC were (93.1 ± 2.3)%, (90.1 ± 3.4)%, (96.2 ± 2.1)% and (98.2 ± 1.7)%, respectively. The average
purity of isolated PC, HSC, SEC, and KC were (98.5 ± 1.2)%, (95.1 ± 2.5)%, (93.6 ± 3.6)% and (98.1 ± 1.4)%,
respectively. HSC underwent a gradual phenotypic transition to a myofibroblast-like phenotype with vitamin
A losing in vitro culture. The apoptosis of SEC began at Day 3, and reached maximum level at Day 7. Then a
few survived SEC started proliferation, and splitted forming a cobblestone, sheet-like appearance at Day 14.
Compared with new isolated SEC, the SEC at Day 14 lost fenestrations, but retained the function of scavenger.

KC could proliferate in vitro which had the ability of phagocytosis, and could express CD 68 (marker of KC)
at Day 14 culture. Conclusions The simultaneous method for isolation was able to obtain the high yield,
viable and purified liver cells from liver of rats, which is useful for the further research of liver physiology
and pathology.