Cultures of primary
hepatocytes from various species, including human, are used in several applications during pre-clinical
drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of
hepatocytes in culture strongly depends on medium formulation and the
extracellular matrix environment. We incubated primary rat
hepatocytes for 10 days on
collagen monolayer and in
collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional
bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or
apoptosis, were less pronounced between alternative cultures. The
intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of
cytochrome P450 enzymes (
P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas
CYP3A showed increased levels in both serum-free cultures. Inducibility of these
P450s was fully maintained over time in serum-free
collagen sandwich only.
Gene expression was largely constant over time in serum-free sandwich cultures that was closest to
liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free
collagen sandwich culture of primary rat
hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term
toxicity and
drug-drug interaction studies.
