Metabolic studies of hepatocytes cultured on collagen substrata modified to contain glycosaminoglycans.

Bioartificial liver devices replace the function of the failing liver, using primary hepatocytes cultured in a bioreactor module. Most devices have been based on cartridge designs, but alternative designs using monolayers of cells in a flat plate bioreactor may be more efficacious. Collagen coating improves the maintenance of hepatocytes on polymeric membranes, and in this article the effect of contact with glycosaminoglycans (GAGs) on the function of hepatocytes was assessed. The effect of two different GAGs, chondroitin-6-sulfate and heparin, in the presence and absence of a cross-linking agent (1,6-diaminohexane [DAH]), on the activities of two major metabolic pathways in hepatocytes (cytochrome P-450-dependent monooxygenase activity, assessed by the hydroxylation of testosterone, and UDP-glucuronosyltransferase activity, assessed by the glucuronidation of kaempferol) cultured on collagen gels and films is presented. Testosterone metabolism was more extensive in cells cultured on collagen films than in cells cultured on gels. The addition of heparin and DAH to collagen gels supported the formation of 6beta-hydroxy, 16alpha-hydroxy, and 2alpha-hydroxy testosterone by cells cultured for 48 h. The extent of glucuronidation of kaempferol was not different when comparing cells cultured on films or gels at the various times in culture; however, the ratio of formation of the two glucuronides formed, M1 and M2, was different. The combination of chondroitin- 6-sulfate and DAH increased glucuronidation of cells cultured for 7 days on both collagen films and gels. This approach may increase the expression of hepatocyte-specific functions in monolayers cultured on membranes in flat plate bioreactors.

[1]  T. Saito,et al.  Albumin synthesis by rat hepatocytes cultured on collagen gels is sustained specifically by heparin. , 1995, Experimental Cell Research.

[2]  M. Grant,et al.  Chondroitin-6-sulphate incorporated into collagen gels for the growth of human keratinocytes: the effect of cross-linking agents and diamines. , 1996, Biomaterials.

[3]  T. Nakamura,et al.  Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes. , 1984, The Journal of biological chemistry.

[4]  J. Wilson,et al.  Alcohol up-regulates UDP-glucuronosyltransferase mRNA expression in rat liver and in primary rat hepatocyte culture. , 2000, Life sciences.

[5]  U. Christians,et al.  Use of organotypical cultures of primary hepatocytes to analyse drug biotransformation in man and animals. , 1994, Xenobiotica; the fate of foreign compounds in biological systems.

[6]  F. Oesch,et al.  Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes. , 1992, Biochemical pharmacology.

[7]  Sangeeta N Bhatia,et al.  Improving the next generation of bioartificial liver devices. , 2002, Seminars in cell & developmental biology.

[8]  Michael J Lysaght,et al.  Tissue engineering: the end of the beginning. , 2004, Tissue engineering.

[9]  E. J. Oliveira,et al.  In vitro glucuronidation of kaempferol and quercetin by human UGT‐1A9 microsomes , 2000, FEBS letters.

[10]  E. Hertzberg,et al.  Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures , 1987, The Journal of cell biology.

[11]  M. P. Arlotto,et al.  Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450. , 1987, Archives of biochemistry and biophysics.

[12]  M. Grant,et al.  The activity of UDP-glucuronyltransferase, sulphotransferase and glutathione-S-transferase in primary cultures of rat hepatocytes. , 1986, Biochemical pharmacology.

[13]  A. Bader,et al.  A Novel Full‐Scale Flat Membrane Bioreactor Utilizing Porcine Hepatocytes: Cell Viability and Tissue‐Specific Functions , 2000, Biotechnology progress.

[14]  E. J. Oliveira,et al.  Metabolism of quercetin and kaempferol by rat hepatocytes and the identification of flavonoid glycosides in human plasma , 2002, Xenobiotica; the fate of foreign compounds in biological systems.

[15]  J. Barbenel,et al.  Investigation into the tensile properties of collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines , 2006, Medical and Biological Engineering and Computing.

[16]  W. Melvin,et al.  Studies on the maintenance of cytochromes P‐450 and b 5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes , 1985, FEBS letters.

[17]  M. Kataropoulou,et al.  The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels , 2003, Human & experimental toxicology.

[18]  K. Jemnitz,et al.  In vitro induction of bilirubin conjugation in primary rat hepatocyte culture. , 2002, Biochemical and biophysical research communications.

[19]  A. Cordier,et al.  Rat adult hepatocytes in primary pure and mixed monolayer culture. Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism. , 1991, Biochemical pharmacology.

[20]  M. Grant,et al.  Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines. , 1999, Biomaterials.

[21]  M. Grant,et al.  Cryopreservation of rat hepatocyte monolayers: cell viability and cytochrome P450 content in post-thaw cultures. , 2002, Toxicology in vitro : an international journal published in association with BIBRA.

[22]  F. Oesch,et al.  Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells. , 1994, Toxicology in vitro : an international journal published in association with BIBRA.

[23]  A. Guillouzo,et al.  Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation. , 1997, Toxicology and applied pharmacology.

[24]  H. Moriwaki,et al.  Regulation of hepatic genes and liver transcription factors in rat hepatocytes by extracellular matrix. , 1995, Biochemical and biophysical research communications.

[25]  Jonathan Bard,et al.  COLLAGEN SUBSTRATA FOR STUDIES ON CELL BEHAVIOR , 1972, The Journal of cell biology.

[26]  Sten Orrenius,et al.  [4] Isolation and use of liver cells , 1978 .

[27]  M. Grant,et al.  Manipulation of the phenotype of immortalised rat hepatocytes by different culture configurations and by dimethyl sulphoxide , 2000, Human & experimental toxicology.

[28]  A. Bader,et al.  Longterm stability of phase I and phase II enzymes of porcine liver cells in flat membrane bioreactors. , 2001, Biotechnology and bioengineering.

[29]  J. Holme,et al.  Drug metabolism activities of isolated rat hepatocytes in monolayer culture. , 2009, Acta pharmacologica et toxicologica.

[30]  R. Tompkins,et al.  Long‐Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration , 1991, Biotechnology progress.

[31]  E. Schuetz,et al.  Hepatocellular phenotype in Vitro is influenced by biophysical features of the collagenous substratum , 1991, Hepatology.

[32]  B J Blaauboer,et al.  The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. , 1990, Biochemical pharmacology.

[33]  E. Hertzberg,et al.  Glycosaminoglycans and proteoglycans induce gap junction expression and restore transcription of tissue‐specific mRNAs in primary liver cultures , 1987, Hepatology.

[34]  A. H. Phillips,et al.  Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. , 1962, The Journal of biological chemistry.