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, allowing to study 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 artefacts or contamination with other cells. The described isolation procedure takes approximately four hours to complete.

Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury

“…We isolated Thy1 MCs as mentioned above from normal mouse liver, and HSCs were subsequently collected from the remaining NPC flow‐through by density‐gradient centrifugation owing to their abundance of retinoid‐storing lipid droplets 25. Isolated Thy1 MCs contained no apparent lipid droplets, which were characteristic of HSCs observed under the phase‐contrast microscope (Fig.…”

Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension

“…Cells were isolated according to Mederacke et al, [30] with the relative modifications for human liver. [31] Briefly, 10 g of total human liver tissue was digested with 0.01% Collagenase, 0.05% Pronase and 0.001% DNase I without performing perfusion.…”

Isolation and Culture of Single Cell Types from Rat Liver

“…However, a systematic procedure for the simultaneous isolation and purification of multiple liver cell populations has only been reported in two previous studies [Liu et al, 2011;Pfeiffer et al, 2014]. Currently, the simultaneous isolation of HCs and NPCs is mainly based on the separation of HCs by low-speed centrifugation [Liu et al, 2011;Pfeiffer et al, 2014] and, as described below, the most common approach to purifying single NPCs is based on cell density [Alpini et al, 1994;Tacke and Weiskirchen, 2012;Mederacke et al, 2015]. The cell densities of LSECs and KCs have some overlap.…”

“…HCs may aggregate with NPCs in subsequent centrifugation steps, thereby increasing the centrifugal density of NPCs and altering the purity and yield of the isolated liver cells [Knook et al, 1982;Alpini et al, 1994]. Therefore, a previous study suggested the administration of nonspecific protease (pronase) through the portal vein during perfusion to selectively lyse HCs for the exclusive isolation of NPCs [Knook and Sleyster, 1976;Alpini et al, 1994;Maschmeyer et al, 2011;Mederacke et al, 2015]. However, this resulted in the loss of HCs.…”