Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors.
Freshly isolated hepatocytes rapidly lose their differentiated properties when placed in culture. Therefore, production of a simple culture system for maintaining the phenotype of hepatocytes in culture would greatly facilitate their study. Our aim was to identify conditions that could maintain the differentiated properties of hepatocytes for up to 28 days of culture. Adult rat hepatocytes were isolated and attached in Williams' medium E containing 10% serum. The medium was changed to either fresh Williams' medium E or keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract. The hepatic phenotype was then analysed using RT-PCR, immunohistochemistry, Western blotting and assays of liver function. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their phenotype for 3-4 weeks, based on expression of liver proteins, ureagenesis and response to xenobiotics. In contrast, hepatocytes cultured in Williams' medium E rapidly lost the expression of liver proteins after 3 days. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their expression of liver-enriched transcription factors (C/EBPalpha and beta, HNF4alpha and RXRalpha) while expression was either lost or reduced in cells cultured in Williams' medium E. These results suggest that keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract can maintain the hepatic phenotype for a prolonged period and that this is probably related to the continued expression of the liver-enriched transcription factors.
Li
Wan-Chun
WC
Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Ralphs
Kate L
KL
Slack
Jonathan M W
JM
Tosh
David
D
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D008112:Liver Glycogen / Q000235:genetics; D019188:Organ Preservation Solutions; D010634:Phenobarbital / Q000494:pharmacology; D010641:Phenotype; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014508:Urea / Q000378:metabolism
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D008112:Liver Glycogen; D019188:Organ Preservation Solutions; D014157:Transcription Factors; C475563:William's medium E; D014508:Urea; D010634:Phenobarbital