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Hepatol Res. 2006 Jul;35(3):169-77. Epub 2006 Jun 27.

An optimal culture condition maintains human hepatocyte phenotype after long-term culture.

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Transplant Research Institute, University of California, Davis Medical Center, Sacramento, CA 95817, USA.



Long-term culture of primary hepatocytes from various species is impeded by a decrease of cell viability and a loss of hepatocyte-specific function. The aim of the present study was to investigate whether our optimal culture condition (OC) can maintain the phenotype of primary hepatocytes in long-term culture.


Primary human hepatocytes were cultured in either hepatocyte maintenance medium (HM) or OC for 2-4 weeks. Expression of hepatocyte-specific genes was determined by real-time quantitative RT-PCR.


The level of albumin mRNA in human hepatocytes cultured in OC was 11-fold more than in HM and gene expression levels of alpha1-antitrypsin and transferrin were at approximately 40 and 11% of freshly isolated primary human hepatocytes. Electron microscopy revealed that cells in OC displayed hepatocyte properties (e.g. polarity, junctional complexes, bile canaliculi and glycogen particles). Cytochrome P4501A1/2 activity of hepatocytes cultured in OC was 15- and 17-fold higher than in HM at 2 and 4 weeks of culture, and DNA synthesis was higher.


Using our optimal culture condition, we were able to maintain the phenotype of primary human hepatocytes in long-term culture. They not only maintain better liver-specific function, but also retain higher proliferative potential.

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