In Vitro Culture of Rat Primary Hepatocytes Employing Monolayer and Spheroid Culture Environments

Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.