CELL MORPHOLOGY, ALBUMIN SECRETION AND GLUTATHIONE-S-TRANSFERASE EXPRESSION IN COLLAGEN GEL SANDWICH AND IMMOBILIZATION CULTURES OF RAT HEPATOCYTES

In order to investigate whether collagen gel sandwich and immobilizati on cultures of rat hepatocytes are suitable in vitro models for long-t erm pharmaco-toxicological studies, the expression of the key phase II biotransformation enzyme, glutathione S-transferase (GST, EC 2.5.1.18 ), has been studied in the presence or absence of L-proline (60 mu g/m l) in the culture medium. Additionally, hepatocytes morphology was fol lowed and albumin secretion into the medium measured. As judged by inv erse phase light microscopy and transmission electron microscopy, cell s cultured in both organotypical models remained viable and well diffe rentiated for at least 14 days. Albumin secretion increased 2.5-fold a fter 7 days of culture, in comparison with the values found after 2 da ys, and remained thereafter relatively constant. When L-proline was ad ded to the medium of sandwich and immobilization gel cultures, steady- state secretion levels of 7.1 and 5.1 mu g albumin/hr, respectively, w ere already obtained after 4 days of culture. Total, Mu, Alpha and Pi class GST activities were determined using a general substrate and iso enzyme specific substrates, respectively. After 7 days of culture, tot al GST activities were decreased as compared with the values obtained for freshly isolated cells. On the contrary, Mu class GST activities w ere kept at a constant level. Alpha class GSTs were maintained at a 50 % activity level and GST 7-7 activity was shown to be slightly induced . L-proline prevented an initial decline in total and Mu class GST act ivities in both culture models. The GST subunit pattern, measured afte r affinity chromatography by reversed phase HPLC, reflected the GST ac tivity results. (C) 1997 Published by Elsevier Science Ltd.