LAUSR

Long‐term functional hepatocyte and reproducible cultures are required in pharmaceutical industries to model chronic liver disorders and to perform associated drug testing. In this frame, we have investigated the behavior of the HepaSH cells when cultivated in liver Biochips, in 3D Spheroids, and in Petri for 20 days. HepaSH is a newly developed humanized hepatocyte harvested from chimeric mice. After the cells' harvesting and inoculation, the HepaSH were successfully maintained in cultures in Petri dishes, spheroids, and Biochips for 20 days. The immunostaining confirmed the expressions of albumin, CYP1A2, and CYP3A4 in all conditions. Furthermore, the CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities were successfully detected in all conditions after 20 days of cultures. Continuous production of albumin and biliary acids was detected in Biochips, Spheroids, and Petri, among which Biochip culture showed the highest albumin secretion level. The RNA sequencing analysis revealed that Biochips and Spheroids cultures enriched hepatic maturation, xenobiotic, lipid, small molecule, steroid, and alcohol metabolisms compared to Petri cultures. Overall, our data demonstrated the feasibility of cultivating the HepaSH cells in Petri, Biochips, and Spheroids for 20 days in the presented protocol, while keeping important liver functions. Biochip and Spheroids cultures show advantages in hepatic maturation, drug metabolism‐related gene expression, and albumin secretion (in biochips) compared with conventional Petri culture.