High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers

Abstract

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.

Figure 1. Isolated HSCs reflect gene expression found in fibrotic livers

Mice were treated with four injections of CCl₄ (0.25 µl/g for the first dose and then 0.5 µl/g i.p. for subsequent doses), dissolved in corn oil at a ratio of 1:3, injected every 3 days. a. Expression of fibrogenic genes Acta2 (encoding for αSMA), Col1a1, Lox and Hhip, (a gene known to be downregulated upon HSC activation), was determined in liver and unplated HSCs isolated by Nycodenz gradient. Gene expression was normalized to 18s. Data are shown as means ± s.e.m. n=5 for control mice and n=10 for CCl₄-treated mice. b. Representative images of freshly isolated HSCs from a control mouse (upper panel) and CCl₄-treated mouse (lower panel) visualized using phase contrast microscopy (left) and retinoid fluorescence (center). A merge (right) of the retinoid fluorescence with the phase contrast image shows complete overlap of retinoid signal with characteristic lipid droplets. Arrows indicate contamination with non-HSC cell types. Scale bars, 50 µm. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University.

Figure 2. Retinoid-based FACS sorting improves purity of HSC isolates without impairing expression of HSC activation markers

a. Retinoid-based ultrapurification of Nycodenz-gradient purified HSCs from untreated (left panel) and CCl₄-treated (right panel) via FACS. b. Gene expression of non-HSC cell contamination markers (Alb for hepatocytes [“Hep“], vWF for liver sinusoid endothelial cells [“LSEC“], Emr1 for liver macrophages [“KC“] and Krt19 for cholangiocytes [“Chol“]) in unsorted, mock-sorted and FACS-ultrapurified HSCs. Relative contamination was determined by comparing the different fractions to pure isolates of hepatocytes, cholangiocytes, liver sinusoidal endothelial cells and liver macrophages (each set as 100% value), respectively. c. HSC activation was determined by qPCR for Col1a1, Acta2, Lox and Hhip in unsorted, mock-sorted and FACS-ultrapurified HSCs. Data are presented as fold induction in comparison to HSCs from control liver. All data are shown as means ± s.e.m. n=5 control mice and n=10 CCl₄-treated mice. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University. ^##p<0.01 and ^### p<0.001 vs. control mock- and unsorted HSCs; * p<0.05 and ** p<0.01 vs. CCl₄-treated mock- and unsorted HSCs; n.s., non-significant.

Figure 3. Overview of HSC isolation procedure

a. Cannulation of anesthetized mouse via IVC (Video 1) and experimental set-up. b. The pump is started, the portal vein is severed and the suprahepatic IVC is clamped (Video 2). c. Sequential perfusion with EGTA, pronase and collagenase solutions. d. The digested liver is excised and minced thoroughly on a Petri dish (Video 3). e. The liver is further digested in vitro and cell-suspension is filtered to remove undigested debris. f. Cells are centrifuged and cell pellet is washed with GBSS/B. g. GBSS/B is overlayed on cell-Nycodenz mixture to create discontinuous gradient (Video 4). h. HSCs are harvested by removing cell layer from the gradient interface (Video 5). i. Final centrifugation to pellet and collect HSCs. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University.