Recent advances in methodology help long-term in vitro maintenance of primary human hepatocytes (PHH), improving their predictability for drug-induced liver injury. Small-molecule additives, such as 5C, have been described to prolong hepatocyte functionality, however, comprehensive description of CYP enzyme and transporter functions are scarce. Here we show a new hepatic model developed by treating hepatocytes in sandwich configuration with 5C (SPHH-5C), where, besides keeping hepatocytes in a fully differentiated form, their polarization is also supported to achieve more in vivo-like structure. The model is characterized by CYP enzyme and hepatobiliary transporter expression and activity as a function of culture time. Hepatic functions were compared in untreated (SPHH) and 5C-treated sandwich (SPHH-5C) or monolayer cultures (PHH-5C). CYP enzyme and transporter functions were best retained in SPHH-5C cultures for 21 days. To simplify the experimental setup and reduce hepatocyte need, a substrate cocktail was applied for simultaneous uptake transporter (OATP1Bs, NTCP, OCT1, OAT2) activity measurements. Beside single donor lots, pooled hepatocyte lots were shown to be applicable for long-term culturing to mitigate interindividual variability. Our novel hepatic model provides a robust and reliable platform for drug screening and safety evaluation by extending in vitro functionality of hepatocytes.
© 2026. The Author(s).