Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

Figure 1

Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation and fluorescence-activated cell sorting (FACS) (schematic depiction). In both strategies for cell purification, the mouse is anaesthetized before surgery, and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of the enzymes pronase and collagenase (a). Liver cells are harvested by gently tearing the liver into bits (b), followed by a postdigestion using a combination of both enzymes (c). The liver cells are subjected to iohexol density gradient centrifugation, after which HSC and Kupffer cells are located in the interphase between iohexol and buffer (d). The enriched HSC layer containing HSC, HSC-Kupffer cell doublets, and cellular debris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS based on the autofluorescence of retinol, using the UV laser of the cell sorter, resulting in highly pure HSC (f).