Formation of porcine hepatocyte spherical multicellular aggregates (spheroids) and analysis of drug metabolic functions

Porcine hepatocytes are used in the hybrid artificial liver support system that we are developing because of their high level of liver functions in vi tro and because human hepatocytes can not be used in Japan for ethical reas ons. Spherical multicellular aggregates or spheroids have been found to be effective in vitro for long-term maintenance of liver functions. Therefore, we formed spherical multicellular aggregates (spheroids) of primary porcin e hepatocytes using a polyurethane foam (PUF) as a culture substratum and a nalyzed their drug metabolic functions in vitro. Primary porcine hepatocyte s inoculated into the pores of a flat PUF plate (25 x 25 x 1 mm), spontaneo usly formed spheroids within the range of 100 to 150 mu m in diameter 24 to 36 h after inoculation. The formed spheroids were attached to the bottom s urface of the PUF pores, and their morphology and viability were maintained for more than 12 days. The P-450 activity in the spheroids of porcine hepa tocytes was demonstrated by detecting production of monoethylglycinexylidid e from lidocaine. In addition, the conjugation enzyme activity was demonstr ated by detecting glucuronidation and sulfation of acetaminophen. These act ivities were maintained for 12 days at a level twice as high as in the mono layer culture. This result shows that the porcine hepatocyte spheroids form ed by using PUF can maintain the drug metabolic functions important in a hy brid artificial liver device. Consequently, culturing porcine hepatocyte sp heroids using PUF seems to be promising for development of a hybrid artific ial liver.