Examination of cytochrome P450 content of primary porcine hepatocyte cultures with various media formulations for use in bioartificial liver devices

Abstract

Primary porcine hepatocytes (PPHs) are used in most bioartificial liver devices (BALs). Liver-specific function, however, deteriorates in these cells in culture. In this study, the PPH biotransformation profile was analyzed over a 6-day culture period, in four separate serum-free media formulations using total cytochrome P450 (P450) content as a marker of differentiation. The general health of the cells in culture was evaluated by analysis of cell morphology, total protein (TP) content, and viability. The total P450 content of bovine adrenocortical cells (BACs) was also evaluated for comparison with hepatocytes. Freshly isolated hepatocyte P450 levels were 120 ± 34 pmol/mg TP, which decreased rapidly in the first 2 days in culture in all media before maintaining P450 levels at between 17 and 28 pmol/mg TP (14%–23% of fresh cell values) at day 6 in culture. Williams' E Medium showed a significantly lower P450 level (25 ± 3 pmol/mg TP) than Hepatocyte Medium (35 ± 3 pmol/mg TP; P = 0.043) on day 4. Williams' E Medium also showed a significantly lower P450 level (17 ± 1 pmol/mg TP) on day 6 than Medium 199 (25 ± 1 pmol/mg TP; P = 0.026) and RPMI-1640 (27 ± 5 pmol/mg TP; P = 0.033). BAC P450 levels decreased to a similar but greater extent to PPHs, decreasing from a fresh cell level of 429 pmol/mg TP to 58 pmol/mg TP (13% fresh cell value) after 6 days in culture. PPHs and BACs show a similar pattern of total P450 decrease in culture. However, Williams' E Medium maintains lower P450 biotransformation potential compared to the other test media studied and is therefore not recommended for use in a BAL.