Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

Figure 5

Hepatic stellate cell activation in vivo and isolation of hepatic stellate cells from livers of injured mice. Chronic toxic liver injury was induced in 8-week-old C57BL/6 mice by 6 weeks of carbon tetrachloride (CCl₄) treatment, and control treatment was done using corn oil. Mice were sacrificed 48 hours after the last injection of (CCl₄) and liver sections were stained for hematoxylin eosin (a), Sirius red (stains collagen fibres, a hallmark of fibrosis) (b), and smooth muscle action (SMA) which targets activated hepatic stellate cells, mediators of fibrosis. Morphometric quantification of Sirius red confirms fibrosis progression (d). Quantitative real-time PCR indicates upregulation of collagen 1 (Col1A1) and SMA mRNA in liver sections (e). Application of the gating strategy of fluorescence-activated cell sorting (FACS) to isolate hepatic stellate cells from livers of mice that underwent six weeks of repetitive CCl₄-based liver injury (f) compared to vehicle corn oil-treated control mice (g). Flow cytometric analysis of HSC purity before and after sorting demonstrated that the purification was successful (h). Mean ± SD of three independent experiments, for control and 16 for fibrotic mice; , , and (unpaired Student’s -test).