Isolation and culture of hepatic stellate cells from mouse liver

Abstract

Hepatic stellate cells (HSCs) are the primary extracellular matrix-producing cells within the liver and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in liver disease. The volume of the mouse liver is much smaller than that of the rat liver, which makes it much more difficult to isolate mouse HSCs (mHSCs) than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with liposome-encapsulated dichloromethylene diphosphate (CL2MDP) to selectively eliminate Kupffer cells from the liver. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution (PBS)-liposome group, the yield of mHSCs was (1.37 ± 0.23) × 10(6)/g liver, the cell purity was (90.18 ± 1.61)%, and the cell survival rate was (94.51 ± 1.61)%. While in the CL2MDP-liposome group, the yield of mHSCs was (1.62 ± 0.34) × 10(6)/g liver, the cell purity was (94.44 ± 1.89)%, and the cell survival rate was (94.41 ± 1.50)%. Based on the yield and purity of mHSCs, the CL2MDP-liposome treatment was superior to the PBS-liposome treatment (P < 0.05, P < 0.01). This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.