Enrichment Methods for Murine Liver Non-Parenchymal Cells Differentially Affect Their Immunophenotype and Responsiveness towards Stimulation

Hepatocytes comprise the majority of the liver and largely exert metabolic functions, whereas non-parenchymal cells (NPCs)-comprising Kupffer cells, dendritic cells and liver sinusoidal endothelial cells-control the immunological state within this organ. Here, we compared the suitability of two isolation methods for murine liver NPCs. Liver perfusion (LP) with collagenase/DNase I applied via the portal vein leads to efficient liver digestion, whereas the modified liver dissociation (LD) method combines mechanical dissociation of the retrieved organ with enzymatic degradation of the extracellular matrix. In cases of both LP and LD, NPCs were enriched by subsequent gradient density centrifugation. Our results indicate that LP and LD are largely comparable with regards to the yield, purity, and composition of liver NPCs. However, LD-enriched liver NPCs displayed a higher degree of activation after overnight cultivation, and accordingly were less responsive towards stimulation with toll-like receptor ligands that are frequently used as adjuvants, e.g., in nano-vaccines. We conclude that LP is more suitable for obtaining liver NPCs for subsequent in vitro studies, whereas LD as the less laborious method, is more convenient for parallel isolation of larger numbers of samples for ex vivo analysis.