Isolation of primary hepatocytes and non-parenchymal cells from mouse fatty liver.

In the early stages of metabolism-associated fatty liver disease (MAFLD), lipid accumulation occurs in the parenchymal cells (hepatocytes, HPCs) of the liver, manifested by an increase in the number of lipid droplets within hepatocytes and an overall decrease in cellular density. Intercellular communication among different liver cell types influences hepatic lipid accumulation, making the impact of non-parenchymal cells (NPCs)on HPCs particularly significant. In vitro HPC models have been well-established and widely applied in MAFLD research. However, the altered microenvironment in MAFLD and cross-contamination between HPCs and NPCs make gene expression research in a single cell type imprecise. To more precisely detect differential gene expression between the two cell types, we enhanced cleaning details during HPC extraction using the perfusion method and adjusted the Percoll concentration gradient to 25%/60% during NPCs extraction. Compared with the previous method, our results show higher cell viability of 94.3% for HPCs and 91.4% for NPCs in the livers of HFD (High Fat Diet) mice. Furthermore, the purity of HPCs and NPCs is higher. This methodological refinement enables accurate quantification of intercellular differences in gene expression, opening new avenues to delineate the role of cell-cell crosstalk in MAFLD pathogenesis.