The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels

M. Kataropoulou, C.J. Henderson, M.H. Grant

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Abstract

    The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture and the inability of the cells to proliferate. The aim of this study was to investigate the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v) and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-S-transferases (GSTs) and the activity of cytochrome P450 in hepatocytes cultured for 48 hours and 7 days. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. Cell homogenates were immuno-blotted against class a and n GST subunits. To measure cytochrome P450 activity, testosterone hydroxylation was assessed. Viability of the cultured cells was assessed by confocal laser scanning microscopy using the vital stain carboxyfluorescein diacetate (CFDA). Cells cultured on gels crosslinked with EDAC were dead by 48 hours as judged by lack of CFDA-derived fluorescence and absence of GST bands on the immunoblots. The viability and morphology of the cells were unaffected by any of the other components of the substrata tested. Expression of GSTs indicated that the hepatocyte phenotype was stable for at least 48 hours. The addition of GAG did not improve the phenotype at either 48 hours or 7 days in culture, but the combination of GAG and DAH crosslinking improved GST expression in the 7-day cultures. However, the hepatocyte cytochrome P450 activity did not show any improvement on any of the gels. The combination of GAG and DAH crosslinking provided the most stable substratum environment in terms of GST expression in hepatocytes.
    Original languageEnglish
    Title of host publicationTransactions of the 25th Annual Meeting of the Society for Biomaterials
    Publication statusPublished - 2001

    Fingerprint

    glycosaminoglycans
    glutathione transferase
    hepatocytes
    collagen
    gels
    phenotype
    crosslinking
    cytochrome P-450
    cultured cells
    viability
    collagenase
    confocal laser scanning microscopy
    hydroxylation
    cells
    xenobiotics
    testosterone
    dyes
    cross-linking reagents
    sulfates
    fluorescence

    Keywords

    • collagen crosslinking
    • cytochrome P450
    • extracellular matrix
    • glutathione-S-transferases
    • glycosaminoglycans
    • primary hepatocyte culture

    Cite this

    Kataropoulou, M., Henderson, C. J., & Grant, M. H. (2001). The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. In Transactions of the 25th Annual Meeting of the Society for Biomaterials
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    author = "M. Kataropoulou and C.J. Henderson and M.H. Grant",
    note = "Katarapoulou Margarita; Henderson, Catherine; Grant, Helen. The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. Human and Experimental Toxicology 2003, vol. 22, no2, pp. 65-71 (This is a variant record)",
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    Kataropoulou, M, Henderson, CJ & Grant, MH 2001, The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. in Transactions of the 25th Annual Meeting of the Society for Biomaterials.

    The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. / Kataropoulou, M.; Henderson, C.J.; Grant, M.H.

    Transactions of the 25th Annual Meeting of the Society for Biomaterials. 2001.

    Research output: Chapter in Book/Report/Conference proceedingChapter

    TY - CHAP

    T1 - The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels

    AU - Kataropoulou, M.

    AU - Henderson, C.J.

    AU - Grant, M.H.

    N1 - Katarapoulou Margarita; Henderson, Catherine; Grant, Helen. The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. Human and Experimental Toxicology 2003, vol. 22, no2, pp. 65-71 (This is a variant record)

    PY - 2001

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    N2 - The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture and the inability of the cells to proliferate. The aim of this study was to investigate the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v) and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-S-transferases (GSTs) and the activity of cytochrome P450 in hepatocytes cultured for 48 hours and 7 days. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. Cell homogenates were immuno-blotted against class a and n GST subunits. To measure cytochrome P450 activity, testosterone hydroxylation was assessed. Viability of the cultured cells was assessed by confocal laser scanning microscopy using the vital stain carboxyfluorescein diacetate (CFDA). Cells cultured on gels crosslinked with EDAC were dead by 48 hours as judged by lack of CFDA-derived fluorescence and absence of GST bands on the immunoblots. The viability and morphology of the cells were unaffected by any of the other components of the substrata tested. Expression of GSTs indicated that the hepatocyte phenotype was stable for at least 48 hours. The addition of GAG did not improve the phenotype at either 48 hours or 7 days in culture, but the combination of GAG and DAH crosslinking improved GST expression in the 7-day cultures. However, the hepatocyte cytochrome P450 activity did not show any improvement on any of the gels. The combination of GAG and DAH crosslinking provided the most stable substratum environment in terms of GST expression in hepatocytes.

    AB - The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture and the inability of the cells to proliferate. The aim of this study was to investigate the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v) and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-S-transferases (GSTs) and the activity of cytochrome P450 in hepatocytes cultured for 48 hours and 7 days. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. Cell homogenates were immuno-blotted against class a and n GST subunits. To measure cytochrome P450 activity, testosterone hydroxylation was assessed. Viability of the cultured cells was assessed by confocal laser scanning microscopy using the vital stain carboxyfluorescein diacetate (CFDA). Cells cultured on gels crosslinked with EDAC were dead by 48 hours as judged by lack of CFDA-derived fluorescence and absence of GST bands on the immunoblots. The viability and morphology of the cells were unaffected by any of the other components of the substrata tested. Expression of GSTs indicated that the hepatocyte phenotype was stable for at least 48 hours. The addition of GAG did not improve the phenotype at either 48 hours or 7 days in culture, but the combination of GAG and DAH crosslinking improved GST expression in the 7-day cultures. However, the hepatocyte cytochrome P450 activity did not show any improvement on any of the gels. The combination of GAG and DAH crosslinking provided the most stable substratum environment in terms of GST expression in hepatocytes.

    KW - collagen crosslinking

    KW - cytochrome P450

    KW - extracellular matrix

    KW - glutathione-S-transferases

    KW - glycosaminoglycans

    KW - primary hepatocyte culture

    UR - http://dx.doi.org/10.1191/0960327103ht320oa

    UR - http://www.esbiomaterials.eu/main/index....

    UR - http://www.biomaterials.org/

    UR - http://strathprints.strath.ac.uk/6769/

    M3 - Chapter

    SN - 8024572001

    BT - Transactions of the 25th Annual Meeting of the Society for Biomaterials

    ER -

    Kataropoulou M, Henderson CJ, Grant MH. The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. In Transactions of the 25th Annual Meeting of the Society for Biomaterials. 2001