Objective: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). Methods: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. Results: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/mug DNA and of 1.8 +/- 0.14 mug/h/mug DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/mug DNA (28.7, 42.8) and 32.7 ng/h/mug DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively, Conclusion: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems. Copyright (C) 2001 S.Karger AG, Basel.