Self-organization of liver constitutive cells mediated by artificial matrix and improvement of liver functions in long-term culture

We previously described the mass preparation of hepatocyte spheroids in a cell suspension. Addition of a synthetic polymer, Eudragit S100 (a co-polymer of methacrylic acid and methylmethacrylate), to the culture medium as an artificial matrix has been found to promote cell aggregation and induce spheroid formation. The present study examines co-cultures of hepatocytes and liver non-parenchymal cells to further enhance the stable expression of liver functions. Hetero-spheroids consisting of hepatocytes and non-parenchymal cells were also formed by adding the polymer to the culture medium. The expression levels of liver functions depended on the mixing ratio of the cells. The values of albumin secretion, ammonia removal and urea synthesis were the highest when equal number of hepatocytes and non-parenchymal cells were mixed. The hetero-spheroids maintained a higher level of albumin secretion for over 4 weeks compared with co-cultured cells without the polymer and spheroids consisting of hepatocytes solely. Other liver functions such as ammonia removal and cytochrome P-450 activity in hetero-spheroid cultures were also maintained at a high level throughout the 4-week culture period.