1P-07 PDF
Analysis of protein expression dynamics in primary cultured mouse hepatocytes
Primary cultures of hepatocytes are widely used as an in vitro model for investigations of hepatic functions, xenobiotic metabolism, and drug toxicity. However, their utility is hindered by alteration of the original hepatocytic phenotype with time. To characterize temporal phenotypic changes in cultures, we examined protein expression dynamics of cultured mouse hepatocytes by shotgun proteomics and semi-quantitative analysis with spectral counting. We used conventional monolayer cultures on type I collagen-coated dishes, as well as spheroidal aggregate cultures on Primaria dishes. Among a total of ~900 unique proteins identified, ~700 proteins could be analyzed semi-quantitatively. After one day in monolayer cultures, the expression of mitochondria-related proteins was increased, while that of metabolic enzymes, such as the cytochrome P450 family, was decreased. Furthermore, expression levels of cytoskeleton-associated proteins were increased with time, apparently correlating with morphological changes. After three days in monolayer cultures, the expression of proteins involved in fatty acid and amino acid metabolism was better preserved as compared with spheroidal cultures. In contrast, proteins involved in glycolysis and gluconeogenesis were expressed at higher levels in spheroidal cultures. Our experiments provide a detailed proteomics data set to direct further works to establish optimum culture methods for mouse hepatocytes.