Cultivation and Characterization of a New Immortalized Human Hepatocyte Cell Line, HepZ, for Use in an Artificial Liver Support System

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Abstract

ABSTRACT: The new human hepatocyte cell line HepZ was investigated with regard to use it for a mass cell cultivation. The cells were originally derived from a human liver biopsy and immortalized through lipofectamine-mediated transfection of albumin-promotor-regulated antisense constructions against the negative controlling cell cycle proteins Rb and p53 (pAlb asRb, pAlb asp53). Furthermore, plasmids including genes coding for the cellular transcription factor E2F and D1 cyclin (pCMV E2F, pSV2neo D1) were cotransfected to overcome the G1-restriction point. Cell cultivation was performed in a 2-liter bioreactor with a working volume of 1 liter. With CultiSpher G microcarriers used in a concentration of 3 g/l a maximal density of 7.1 3 106 cells/ml was achieved in a cultivation period of 20 days. The cells exhibited a maximal specific growth rate of 1.0 per day in the first 4 days. After 9 days of cultivation the stationary growth phase was reached with an average cell density of 5.5 3 106 cells/ml. The viability status of the culture was determined indirectly by measuring of the lactate dehydrogenase activity (LDH) at 378C. During the growth phase the activity rose slightly up to a value of 200 U/l. The cells were flat after first attachment on the gelatine microcarriers and spherical after growing into the three-dimensional inner matrix-both of which characteristics were verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The liver-specific cytochrome P450 activity was challenged with a pulse of 7 mg/ml lidocaine at a cell density of 4.5 3 106 cells/ml. After an induction period of 3 days with 50 μg/ml of phenobarbital, 26 ng/ml MEGX were generated within one day compared to 5 ng/ml without induction. The new cell line HepZ has proven to retain liver-specific qualities and to be appropriate for mass cell cultivation for bioartificial devices.

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