Effects of cell culture conditions on primary rat hepatocytes-cell morphology and differential gene expression.

We incubated primary rat hepatocytes on collagen monolayer as well as in collagen sandwich cultures with serum-containing or serum-free medium formulations. Morphological monitoring of hepatocytes revealed that hepatocytes cultured on collagen monolayer adopted their polygonal shape and started to create aggregates earlier than sandwich-cultured cells. Bile canaliculi-like structures were observed in every cell culture system but were more prominent in serum-free cultures. Hepatocytes in collagen-sandwich configuration and serum-free medium were the most viable after 72 h of culture, still displaying polygonal shape, clear cytoplasm and stable canaliculi-like structures. Differential gene expression patterns were determined for each cell culture condition using quantitative TaqMan Low Density Arrays (LDA). Gene expression analysis revealed distinct profiles in monolayer versus sandwich cultures and in particular in serum-free versus serum-containing culture medium. The hepatocytes cultured in the collagen-sandwich with serum-free medium showed the least variation in expression values over time. Importantly, stress markers were not induced in the serum-free sandwich culture, in contrast to the monolayer and the serum-containing sandwich cultures. Additionally, expression of the investigated cytochrome P450 genes was maintained in the serum-free monolayer and the sandwich cultures. In conclusion, culturing primary rat hepatocytes in a sandwich between two layers of gelled collagen and in a serum-free medium formulation, appears to be most suitable for long-term in vitro hepatotoxicity screening.

[1]  A. Y. Lu,et al.  N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Emery,et al.  Unique gene expression patterns in liver and kidney associated with exposure to chemical toxicants. , 2001, The Journal of pharmacology and experimental therapeutics.

[3]  D. Miner,et al.  Evidence for the involvement of N-acetyl-p- quinoneimine in acetaminophen metabolism. , 1979, Biochemical pharmacology.

[4]  K. Audus,et al.  Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. , 1994, The American journal of physiology.

[5]  P. Seglen.  Preparation of isolated rat liver cells. , 1976, Methods in cell biology.

[6]  W. Melvin,et al.  Studies on the maintenance of cytochromes P‐450 and b 5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes , 1985, FEBS letters.

[7]  Chow H Lee.  Differential regulation of P‐glycoprotein genes in primary rat hepatocytes by collagen sandwich and drugs , 2002, Journal of cellular biochemistry.

[8]  J. S. Sidhu,et al.  Phenobarbital responsiveness as a uniquely sensitive indicator of hepatocyte differentiation status: requirement of dexamethasone and extracellular matrix in establishing the functional integrity of cultured primary rat hepatocytes. , 2004, Experimental cell research.

[9]  W. Gallin,et al.  Effects of fetal calf serum and disruption of cadherin function on the formation of bile canaliculi between hepatocytes. , 1994, Experimental cell research.

[10]  R. Tompkins,et al.  Long‐Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration , 1991, Biotechnology progress.

[11]  P. Guzelian,et al.  PHENOTYPIC STABILITY OF ADULT RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE * , 1980, Annals of the New York Academy of Sciences.

[12]  D. Jefferson,et al.  Culturing hepatocytes and other differentiated cells , 1984, Hepatology.

[13]  O. Fardel,et al.  Regulation by dexamethasone of P‐glycoprotein expression in cultured rat hepatocytes , 1993, FEBS letters.

[14]  G. Williams,et al.  Rat hepatocyte primary cell cultures , 1977, In Vitro.

[15]  E Dybing,et al.  Cytotoxic effects of N-acetyl-p-benzoquinone imine, a common arylating intermediate of paracetamol and N-hydroxyparacetamol. , 1984, Biochemical pharmacology.

[16]  J. S. Sidhu,et al.  Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture. , 1993, Archives of biochemistry and biophysics.

[17]  Andrew Parkinson,et al.  Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes , 1996 .

[18]  Thierry Arnould,et al.  Use of a low-density microarray for studying gene expression patterns induced by hepatotoxicants on primary cultures of rat hepatocytes. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[19]  J. S. Sidhu,et al.  Modulation of xenobiotic-inducible cytochrome P450 gene expression by dexamethasone in primary rat hepatocytes. , 1995, Pharmacogenetics.

[20]  Lee Bennett,et al.  Prediction of compound signature using high density gene expression profiling. , 2002, Toxicological sciences : an official journal of the Society of Toxicology.

[21]  Gordon Vansant,et al.  Gene expression profiling of rat livers reveals indicators of potential adverse effects. , 2004, Toxicological sciences : an official journal of the Society of Toxicology.

[22]  E. Santoni-Rugiu,et al.  Differential modulation of P-glycoprotein expression by dexamethasone and 3-methylcholanthrene in rat hepatocyte primary cultures. , 1994, Carcinogenesis.

[23]  H. Pitot,et al.  Reestablishment of cell polarity of rat hepatocytes in primary culture , 1993, Hepatology.

[24]  D. Waxman,et al.  Impact of dimethyl sulfoxide on expression of nuclear receptors and drug-inducible cytochromes P450 in primary rat hepatocytes. , 2004, Archives of biochemistry and biophysics.

[25]  R. Tompkins,et al.  Hepatocyte function and extracellular matrix geometry: long‐term culture in a sandwich configuration , 1989, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.