[An improved method for the isolation of rat hepatic stellate cells].

OBJECTIVE

To present an improved method to obtain pure, viable, freshly isolated hepatic stellate cells.

METHODS

Adult male SD rats were used. All procedures were performed with the animals under sodium pentobarbital anesthesia. Three days after the single intravenous administration of 1 ml liposome-encapsulated CL2MDP, which has selective cytotoxicity of Kupffer cells, livers were perfused with D-Hank's solution containing 100 U/ml heparin for 10 to 15 minutes, and then with 0.05% collagenase dissolved in D-Hank's solution for 25 to 30 minutes. The liver was then gently homogenized and further incubated in 0.025% collagenase, and 0.005% DNAase I for 30 minutes at 37 degrees C under constant stirring. This suspension was filtered through stainless steel gauze and centrifuged for 2 minutes at 50 x g to remove parenchymal cells. Sinusoidal cells in the supernatant were recovered by centrifugation for 10 minutes at 300 x g. The cells were resuspended in the presence of 28.7% Nycodenz stock solution. The final concentration of Nycodenz at this stage was 11.5%. Following centrifugation for 17 minutes at 1400 x g, The cells at the top of this Nycodenz solution were collected. Cells were resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, The cells were seeded in 50 ml culture flask at a density of 500,000 cells/ml, The cell viability was determined by trypan blue exclusion staining, the purity of hepatic stellate cells was identified by the expression of Desmin using immunocytochemistry method. Endogenous peroxidase staining was used to detect Kupffer cells.

RESULTS

The yield rate of hepatic stellate cells was 3 x 10(7) per rat, the cell viability was more than 95%, the desmin positive cell rate was 90%, no endogenous peroxidase positive cells were detected.

CONCLUSION

The method for the isolation of hepatic stellate cells was developed without Kupffer cells confusion. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.