Premium
Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers
Author(s) -
Werner Andreas,
Duvar Sevim,
Müthing Johannes,
Büntemeyer Heino,
Lünsdorf Heinrich,
Strauss Michael,
Lehmann Jürgen
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000405)68:1<59::aid-bit7>3.0.co;2-n
Subject(s) - microcarrier , bioreactor , laboratory flask , bioartificial liver device , chromatography , chemistry , cell culture , hepatocyte , aeration , microbiology and biotechnology , cell , biology , biochemistry , in vitro , genetics , organic chemistry
Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 × 10 5 cells/mL when using a microcarrier concentration of 1 g/L and 5 × 10 5 cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 × 10 6 cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble‐free aeration and daily medium exchange from day 7, 7.1 × 10 6 cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat‐shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 m M in the spinner culture and up to 9.1 m M in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10 6 cells·day). HepZ cells resisted a 4‐day long chilling period at 9.5°C. The cytochrome P450 system was challenged with a pulse of 7 μg/mL lidocaine at a cell density of 4.5 × 10 6 cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 μg/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 59–70, 2000.