Hepatocytes are cultured in many formats. For short term metabolic assays (less than 4 hours), hepatocytes can be placed into a reaction vessel in suspension either directly following isolation or after reanimation from cryopreservation.
Hepatocytes are used in plated formats when drugs are poorly metabolized in short-term suspension assays, or when mechanistic data regarding drug-drug interactions and mechanistic toxicity is needed. Hepatocytes plated in a monolayer on a collagen matrix have an extended lifespan compared to cells in suspension and maintain their metabolic function for longer.
In vivo, hepatocytes have a polar morphology whereby cell surface proteins vary on the different 3D surfaces. This polar morphology can be replicated in vitro by providing an overlay of collagen or other basement membrane extracts on top of a hepatocyte monolayer. By “sandwiching” hepatocytes between these basement membrane protein mixtures, they form the correct basal surface structures and a small apical membrane pocket between cells known as bile canaliculi. These structures enable excretion of bile acids from the hepatocytes, replicating the situation in vivo. It is generally accepted that the sandwich culture method reflects the 3D in vivo shape of hepatocytes, while maintaining the functionality of a 2D format.
An emerging technique for recapitulating the in vivo physiology of the hepatocyte is to allow the cells to self-assemble into a spheroid shape. Using simple to use low attachment, round-bottom culture plates, hepatocytes adhere to each other more readily and over the course of 4-5 days will become a tight ball of cells which demonstrate polarity and tight junctions. These small spheroids with about 1500-3000 cells have been shown to function more like the in vivo liver tissue and be better predictors of chemical toxicity that standard hepatocyte monolayer cultures.