Search

Abstract: The use of exogenous matrices has been described as an essential component in securing the viability and functionality of hepatocytes in vitro whether cultured for extracorporeal devices or cell transplantation. Here we report on the in vitro culture of porcine hepatocytes in polystyrene tissue-culture flasks without exogenous matrices showing adequate attachment and viability. Cell proliferation was evidenced by uptake of 5-bromo-2′-deoxyuridine, with peaks at Days 2 (19.7 ± 8.5%), 15 (20.8 ± 3.3%), and 35 (21.4 ± 0.3%). Detoxification capacity was assessed by determination of monoethylglycinexylidide, a product of lidocaine metabolism (highest value 156.5 ± 10.1 ng/ml at Day 4), and by diazepam clearance (maximum clearance 66.2% at Day 6). Diazepam metabolite levels were highest at Day 4 both for temazepam and oxazepam (6.5 ± 0.1 and 0.10 ± 0.01, respectively). These results suggest that the need for an exogenous matrix to achieve sustained proliferative activity and differentiated hepatocyte function should not necessarily be considered a sine qua non condition. [ABSTRACT FROM AUTHOR]

Porcine hepatocytes are used in the hybrid artificial liver support system that we are developing because of their high level of liver functions in vitro and because human hepatocytes can not be used in Japan for ethical reasons. 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 porcine hepatocytes using a polyurethane foam (PUF) as a culture substratum and analyzed their drug metabolic functions in vitro. Primary porcine hepatocytes inoculated into the pores of a flat PUF plate (25 × 25 × 1 mm), spontaneously formed spheroids within the range of 100 to 150 μm in diameter 24 to 36 h after inoculation. The formed spheroids were attached to the bottom surface 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 hepatocytes was demonstrated by detecting production of monoethylglycinexylidide from lidocaine. In addition, the conjugation enzyme activity was demonstrated by detecting glucuronidation and sulfation of acetaminophen. These activities were maintained for 12 days at a level twice as high as in the monolayer culture. This result shows that the porcine hepatocyte spheroids formed by using PUF can maintain the drug metabolic functions important in a hybrid artificial liver device. Consequently, culturing porcine hepatocyte spheroids using PUF seems to be promising for development of a hybrid artificial liver. [ABSTRACT FROM AUTHOR]