Efficient Formation of Three Dimensional Spheroids of Primary Hepatocytes Using Micropatterned Multi-Well Plates

Primary hepatocytes constitute 60-80% of liver where they play a vital role in metabolism, synthesis and storage such as vitamins, carbohydrates, proteins. When hepatocytes are grown in vitro as monolayers, they rapidly lose their phenotypic characteristics and functional abilities. We successfully developed a threedimensional (3D) culturing system for rat primary hepatocytes using micropatterned multi-well plates. The efficiency, morphology, cell viability and functionality (albumin secretion and urea synthesis) of hepatocyte spheroids produced by 3D and monolayer culture were compared. In monolayer culture, hepatocytes did not maintain an intact morphology and their viabilities started to decline after 4 days. On the other hand, hepatocyte spheroids were prepared with various numbers of single hepatocytes. It was confirmed that 150 hepatocytes per spheroid were optimum with incorporation efficiencies of around 95% as well as obtained spheroids were more homogenous in shape and size. Additionally, the secretion of albumin and production of urea by these spheroids were significantly greater than those of cells in monolayer culture. Our results show the described method could potentially be used in liver tissue regeneration, new drug discovery or toxicology studies.