Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix.
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).
Gómez-Lechón
M J
MJ
Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain. mjgomez@san.gva.es
Jover
R
R
Donato
T
T
Ponsoda
X
X
Rodriguez
C
C
Stenzel
K G
KG
Klocke
R
R
Paul
D
D
Guillén
I
I
Bort
R
R
Castell
J V
JV
D000818:Animals; D001711:Biotransformation; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen; D016895:Culture Media, Serum-Free; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D004790:Enzyme Induction / Q000187:drug effects; D005109:Extracellular Matrix; D005786:Gene Expression Regulation; D016762:Genes, fos; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008112:Liver Glycogen / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D004364:Pharmaceutical Preparations / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014508:Urea / Q000378:metabolism
D016895:Culture Media, Serum-Free; D007328:Insulin; D007527:Isoenzymes; D008112:Liver Glycogen; D004364:Pharmaceutical Preparations; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D014157:Transcription Factors; D014508:Urea; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System