Due to the different geometry, the system shook on a Plate (Orbit 3 mm) at 150 rpm , while the Petri dishes were shaken on an orbital (orbit 20 mm) at 50 rpm. found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or RICTOR NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a gold standard culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to hepatic tissue formation. for pharmacological research and hepatocyte research, including bioartificial liver supports. Primary hepatocyte cells are always preferable, as these cells closely mimic the in vivo state and generate more physiologically relevant data than cell lines. culture of primary BACE1-IN-4 hepatocytes is a useful model for the expression and regulation of liver genes . However, the main disadvantage is that primary cells lose their state of metabolic function in the conventional monolayer due to the lack of a proper multicellular three-dimensional microenvironment like polarity of liver architecture. Under some circumstances, unattached hepatocytes generally do self-assemble into multicellular spheroids. Mature hepatocyte spheroid culture models are similar to a 3D culture model with improved cellCcell and cellCmatrix interactions; they also display higher levels of liver-specific functions, such as BACE1-IN-4 high cytochrome P450 activity , albumin production [3,4,5,6,7,8], long-term culture up to 60 days transferrin secretion , ureagenesis , and tyrosine aminotransferase induction , than are displayed in monolayer cultures. Such a 3D culture model has occurred to recapitulate many in vivo tissue structures and functions [3,9]. Very few hepatocyte spheroid models were established using: a poly-(L-lactic acid ) polymer , rock techniques , micro-rotation flows , alginate scaffolds , RGD and galactose-conjugated membranes , positive-charged substrates , micropatterning techniques , nanopillar sheets , galactosylated nanofiber scaffold , or polyurethane forms . However, hepatocyte spheroids under the influence of fetal calf serum and nonparechyalmal cells have not yet been established. Since 3D polarity is a vital and typical property of hepatocytes and necessary for proper hepatic functions, this present study attempted to create a multicellular spheroid on a poly-(HEMA)-treated surface under influence of fetal calf serum and nonparechyalmal cells. Sandwich-cultured hepatocytes are a promising cellular model . In our previous study, the rates of metabolite formation are much lower in conventional primary hepatocyte culture models than in the organotypical model . The sandwich culture model enables the conservation of liver-specific characteristics such as cuboidal morphology of hepatocytes, bile canaliculi, tight junctions, and gap junctions [21,22,23,24,25,26]. Furthermore, we recently reported on two compartment models of biotransformation BACE1-IN-4 of the drug diazepam in primary human hepatocytes to show that the metabolites of diazepam are present in two compartments (collagen matrix and supernatant with drugCdrug interaction in an organotypical model . BACE1-IN-4 However, the sandwich model is a well-accepted model for wide varieties of hepatic tissue engineering, including bioartificial liver devices [28,29], toxicology studies . Lee  recently report that the hepatocyte spheroid-based BAL system may be BACE1-IN-4 a noble nominee for treatment of liver failure patients. Furthermore, isolated hepatocytes are not able to maintain the cell membrane polarity  whereas hepatocytes.