Rat hepatocyte spheroids formed on temperature-responsive PIPAAm polymer-grafted surface maintain long-term differentiated hepatocyte function

Introduction The development of effective culture methods is very important in regenerative medicine. This is because they may be able to contribute to the advancement of medicine. Since the liver plays an important and complex metabolic role, the development of a practicable culture method to maintain long-term hepatocyte function is highly desirable. However, hepatocytes lose their liver-specific functions rapidly on a regular collagen-coated dish (1). Developing an incubation system which can maintain the liver function would markedly contribute to regenerative medicine. Recently, a newly developed culture dish was introduced coated with the temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) (2-9), whereby temperature fluctuations can mediate changes in the chemistry of the Background. Although the development of a practicable culture method to maintain long-term hepatocyte function is highly desirable, hepatocytes lose their liver-specific functions rapidly on regular collagen-coated dishes. In this study, we evaluated the efficacy of a special cell culture dish coated with the temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm), and observed the morphological and functional changes of hepatocytes on changing the amount of polymer. Methods. Culture plates with varying amounts of PIPAAm polymer (1.5〜3.5 times that in the commercially available temperature-responsive polymer-containing dish (UpCell ® , CellSeed, Tokyo, Japan)) were prepared. All dishes were enhanced by adding a layer of rat tail collagen I at a dose of 600 µg/dish for 3 hours. Hepatocytes isolated from Sprague-Dawley rats were cultured in these static culture systems. Morphologic changes and liver-specific functions were evaluated. Results. On day 7 of culture, spheroid formation of hepatocytes was observed in the high-polymer group, with the presence of gly-cogen and albumin in the spheroid. In the high-polymer group, the rate of albumin production was significantly higher than in the low-polymer group until day 14 of culture (P<0.001). Conclusion. The spheroid formation of hepatocytes cultured in the presence of a high level of PIPAAm showed the long-term maintenance of liver-specific functions in vitro.