Effects of cell culture conditions on primary rat hepatocytes—Cell morphology and differential gene expression

Abstract

We incubated primary rat hepatocytes on collagen monolayer as well as in collagen sandwich cultures with serum-containing or serum-free medium formulations. Morphological monitoring of hepatocytes revealed that hepatocytes cultured on collagen monolayer adopted their polygonal shape and started to create aggregates earlier than sandwich-cultured cells. Bile canaliculi-like structures were observed in every cell culture system but were more prominent in serum-free cultures. Hepatocytes in collagen-sandwich configuration and serum-free medium were the most viable after 72 h of culture, still displaying polygonal shape, clear cytoplasm and stable canaliculi-like structures. Differential gene expression patterns were determined for each cell culture condition using quantitative TaqMan ® Low Density Arrays (LDA). Gene expression analysis revealed distinct profiles in monolayer versus sandwich cultures and in particular in serum-free versus serum-containing culture medium. The hepatocytes cultured in the collagen-sandwich with serum-free medium showed the least variation in expression values over time. Importantly, stress markers were not induced in the serum-free sandwich culture, in contrast to the monolayer and the serum-containing sandwich cultures. Additionally, expression of the investigated cytochrome P450 genes was maintained in the serum-free monolayer and the sandwich cultures. In conclusion, culturing primary rat hepatocytes in a sandwich between two layers of gelled collagen and in a serum-free medium formulation, appears to be most suitable for long-term in vitro hepatotoxicity screening.