Recent animal studies and clinical trials using bioartificial liver devices have shown great promise for the treatment for acute liver failure, and are providing valuable information on the problems and limitations of current ''1st generation" liver assist devices. It is becoming clearer every day, however, that more basic information of the effect of environmental parameters on hepatocellular function, as well as host-bioartificial liver interactions, is necessary before the concept of bioartificial liver becomes a reality available at reasonable cost. Our long-term objective is to help development of 2nd and 3rd generation devices, which are expected to be significantly more effective than currently available devices. In order to reach this goal, we require a better understanding of many critically important questions including: What is the minimum cell mass to support a patient? How long and well do hepatocytes function during plasma exposure? What are the most critical functions for patient survival? What is the impact of bioartificial liver treatment on the immune system and on subsequent liver transplantation? Answers to these questions will often not be obtainable using off-the-shelf tools, and will require the development of new experimental systems. Our main hypothesis is that there is a finite number of hepatic functions which are most critical for patient survival, that it is possible to significantly upregulate them in hepatocyte cultures (both at the single cell level and at the level of tissue), and as a result, reduce the cell mass required in the bioartificial liver. The specific aims are: (1) To use metabolic and genetic engineering approaches to enhance the performance of hepatocyte cultures in plasma; (2) To optimize the oxygenation and geometric configuration of hepatocyte co-cultures for plasma detoxification; (3) To investigate patient-bioartificial liver interactions and characterize the immunological response to extracorporeal perfusion with allo- and xenogeneic cells. These studies will provide the basic knowledge and technologies enabling us to develop the next generation of liver assist devices and speed up the translation of this promising modality to the bedside. The proposed studies will also provide basic tools useful in the development of other engineered tissues and organs.