TY - JOUR
T1 - The influence of glycosaminoglycans and cross-linking agents on the phenotype of hepatocytes cultured on collagen gels
AU - Kataropoulou, M.
AU - Henderson, C.J.
AU - Grant, M.H.
N1 - Also published in Transactions of the 25th Annual Meeting of the Society for Biomaterials. Society for Biomaterials, 2001. ISBN 8024572001
PY - 2003
Y1 - 2003
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-di methylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-Stransferases (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 immunoblotted against class {alpha} and {pi} 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-di methylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-Stransferases (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 immunoblotted against class {alpha} and {pi} 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
KW - extracellular matrix
KW - glutathione-S-transferases
KW - glycosaminoglycans
KW - primary hepatocyte
KW - bioengineering
UR - http://dx.doi.org/10.1191/0960327103ht320oa
U2 - 10.1191/0960327103ht320oa
DO - 10.1191/0960327103ht320oa
M3 - Article
SN - 0960-3271
VL - 22
SP - 65
EP - 71
JO - Human and Experimental Toxicology
JF - Human and Experimental Toxicology
IS - 2
ER -