Hepatocyte engineering. The development of a bioartificial liver

The use of hepatocytes in an extracorporeal liver device is a promising approach for the treatment of liver failure. However, the lack of adequate in vitro culture methods for hepatocytes has been a major obstacle in the development of an effective bioartificial liver device. It is the purpose of this study to engineer optimal culture conditions for rat hepatocytes. We have demonstrated that hepatocytes sandwiched between two layers of collagen maintained morphology and function for a period up to 8 weeks. Differentiated function was also correlated with distribution of actin filaments, extracellular matrix secretion, and membrane protein localization similar to that seen in intact liver. In addition, we examined the effect of different culture media on hepatocyte function under static culture conditions. Albumin secretion rate of hepatocytes cultured in Williams-E medium had better longevity and reached steady-state rate of albumin secretion faster than hepatocytes cultured in Dulbecco's Modified Eagle Medium. The steady-state albumin secretion (5μg/h/10⁶ cells) was comparable to published in vivo values. Most recently, we have examined long-term hepatocyte function in a continuous flow system. In preliminary bioreactor studies, albumin secretion and oxygen consumption rates were measured for a period of 2 weeks. Albumin secretion rates in the bioreactor was comparable to the static culture control. Oxygen uptake rate was stable between 0.5 and 1 mmHg/min/10⁶ cells during the two weeks of culture. These preliminary results suggest that hepatocytes can be cultured successfully in between two layers of collagen under either static or continuous flow conditions.