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 liposomeencapsulated 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) 3 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) 3 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.

MALAT1 functions as a competing endogenous RNA to mediate Rac1 expression by sequestering miR-101b in liver fibrosis

“…43 After the resulting cell suspension was centrifugated at 40 g for 5 min, the pellet was further centrifugated at 25 g for 5 min and the obtained cells were primary hepatocytes. The supernatant was further centrifuged at 400 g for 10 min.…”

HOTAIR Epigenetically Modulates PTEN Expression via MicroRNA-29b: A Novel Mechanism in Regulation of Liver Fibrosis

“…39 The isolated cells were seeded in tissue culture plates and cultured in DMEM (Gibco) with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. The purity of cultures was confirmed by immunocytochemical staining for a-SMA and the purity reached >98%.…”

LincRNA-p21 Inhibits the Wnt/β-Catenin Pathway in Activated Hepatic Stellate Cells via Sponging MicroRNA-17-5p

“…Primary HSCs were isolated from male C57BL/6J mice as described previously [16]. Mice were provided by the Experimental Animal Center of Wenzhou Medical University and the experimental protocol was approved by the Institutional Animal Committee of Wenzhou Medical College.…”