Culturing of primary mouse hepatocytes is associated with major changes to the proteome and transcriptome

Primary hepatocytes are a commonly used in vitro model for studying liver metabolism, but prolonged culturing results in dedifferentiation. Here we characterized the transcriptome and proteome of whole liver and primary hepatocytes as either freshly isolated cells or after 24 hours of 2D-culturing. We found that after 24 hours of 2D-culturing, hepatocytes showed differential expression of over 10,000 genes and 3,000 proteins compared to freshly isolated cells. This was accompanied by a reduced transcriptional heterogeneity and a loss of zonal markers. Cultures contained ~10% non-hepatocytes, including stellate- and dedifferentiated endothelial cells. Culturing also increased abundance of proteins associated with oxidative stress and inflammation, and changed mitochondrial, extracellular, and ribosomal protein abundance. The data generated are available in the shiny-app “Hepamorphosis, https://cbmr-rmpp.shinyapps.io/hepamorphosis/”. Our finding show that primary mouse hepatocytes undergo significant changes in culture, limiting their utility for studying physiological and molecular mechanisms related to the liver. Overall design: Single nuclei sequencing from liver biopsies (L), freshly isolated liver cells in suspension (CS), and primary hepatocytes following 24 hours of culture on collagen (PH)