High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices

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

Pixel and Voxel Representations of Graphs

We study contact representations for graphs, which we call pixel representations in 2D and voxel representations in 3D. Our representations are based on the unit square grid whose cells we call pixels in 2D and voxels in 3D. Two pixels are adjacent if they share an edge, two voxels if they share a face. We call a connected set of pixels or voxels a blob. Given a graph, we represent its vertices by disjoint blobs such that two blobs contain adjacent pixels or voxels if and only if the corresponding vertices are adjacent. We are interested in the size of a representation, which is the number of pixels or voxels it consists of. We first show that finding minimum-size representations is NP-complete. Then, we bound representation sizes needed for certain graph classes. In 2D, we show that, for $k$-outerplanar graphs with $n$ vertices, $\Theta(kn)$ pixels are always sufficient and sometimes necessary. In particular, outerplanar graphs can be represented with a linear number of pixels, whereas general planar graphs sometimes need a quadratic number. In 3D, $\Theta(n^2)$ voxels are always sufficient and sometimes necessary for any $n$-vertex graph. We improve this bound to $\Theta(n\cdot \tau)$ for graphs of treewidth $\tau$ and to $O((g+1)^2n\log^2n)$ for graphs of genus $g$. In particular, planar graphs admit representations with $O(n\log^2n)$ voxels

Hepatic levels of bile acids in end-stage chronic cholestatic liver disease

In chronic cholestatic liver disease hydrophobic and potentially cytotoxic bile acids are assumed to accumulate in the liver. To test this hypothesis we investigated bile acid levels and pattern in livers and serum of patients with, (A) end-stage chronic cholestatic liver disease, and with (B) end-stage cirrhosis of alcoholic/chronic hepatitic origin who underwent liver transplantation. Bile acids were also analyzed in (C) normal liver tissue. Levels of bile acids were 215 +/- 39.1 nmol/g liver (wet weight) in chronic cholestasis and 120 +/- 32.7 and 56.1 +/- 24.2 nmol/g liver in group B and group C (P <0.01 and P <0.005), respectively. Cholic acid was the prevailing bile acid in chronic cholestasis (51%) and was elevated eight-fold as compared to group C (P <0.005). Chenodeoxycholic acid contributed 41% to total bile acids and was elevated four-fold (P <0.005). Deoxycholic acid contributed only 1.5% to bile acids in chronic cholestasis as compared to 27% in group C (P <0.01) and was absent in group B. Levels of lithocholic acid tended to be increased in chronic cholestasis as compared to group C and its sulfation was impaired (P <0.05). The pattern of serum bile acids in chronic cholestasis agreed well with the bile acid pattern in the explanted livers. We conclude that hepatic accumulation of hydrophobic chenodeoxycholic acid and impaired sulfation of lithocholic acid might contribute to tissue degeneration in chronic cholestatic liver disease due to the detergent effects of these bile acid

Perfusion culture improves the maintenance of cultured liver tissue slices

10.1089/ten.2006.0046Tissue Engineering131197-20

Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes

Ursodeoxycholic acid (UDCA) improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. UDCA is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2+ agonist and stimulates biliary exocytosis and hepatocellular Ca2+ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accummulation of sn-1,2-diacylglycerol (DAG), the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. Immunoblotting studies showed the presence of four isoenzymes (alpha, delta, epsilon, and zeta). The phorbol ester phorbol 12-myristate 13-acetate (1 mumol/L) induced translocation of alpha-PKC, delta-PKC, and epsilon-PKC from cytosol to a particulate membrane fraction, a key step for activation of PKC. TUDCA, but not taurocholic acid, selectively induced translocation of the alpha-PKC isoenzyme from cytosol to the membranes. In addition, TUDCA induced a significant increase in hepatocellular DAG mass and stimulated membrane-associated PKC activity. TUDCA might stimulate Ca(2+)-dependent hepatocellular exocytosis into bile in part by activation of alpha-PK