title: The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels.
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 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.
authors.affiliation: Bioengineering Unit, Wolfson Centre, University of Strathclyde, 106 Rottenrow, Glasgow G4 0NW, UK.
authors.forename: Margarita
authors.initials: M
authors.lastname: Kataropoulou
authors.forename: Catherine
authors.initials: C
authors.lastname: Henderson
authors.forename: Helen
authors.initials: H
authors.lastname: Grant
mesh_terms: D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000494:pharmacology*; D003094:Collagen / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003959:Diamines / Q000494:pharmacology*; D004338:Drug Combinations; D005782:Gels; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003470:Culture Media; D003959:Diamines; D004338:Drug Combinations; D005782:Gels; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D002809:Chondroitin Sulfates; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; C007297:1,6-diaminohexane