Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix

J Cell Physiol. 1998 Dec;177(4):553-62. doi: 10.1002/(SICI)1097-4652(199812)177:4<553::AID-JCP6>3.0.CO;2-F.


Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotransformation
  • Cell Culture Techniques / methods*
  • Cell Differentiation
  • Cells, Cultured
  • Collagen
  • Culture Media, Serum-Free
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / physiology
  • Enzyme Induction / drug effects
  • Extracellular Matrix
  • Gene Expression Regulation
  • Genes, fos
  • Insulin / pharmacology
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics
  • Isoenzymes / physiology
  • Liver / cytology*
  • Liver / metabolism
  • Liver Glycogen / metabolism
  • Male
  • Microsomes, Liver / enzymology
  • Pharmaceutical Preparations / metabolism
  • Proto-Oncogene Proteins c-fos / biosynthesis
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics
  • Urea / metabolism


  • Culture Media, Serum-Free
  • Insulin
  • Isoenzymes
  • Liver Glycogen
  • Pharmaceutical Preparations
  • Proto-Oncogene Proteins c-fos
  • RNA, Messenger
  • Transcription Factors
  • Urea
  • Collagen
  • Cytochrome P-450 Enzyme System