Time-related analysis of metabolic liver functions, cellular morphology, and gene expression of hepatocytes cultured in the bioartificial liver of the Academic Medical Center in Amsterdam (AMC-BAL).
A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.
Poyck
Paul P C
PP
Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
Hoekstra
Ruurdtje
R
Chhatta
Aniska
A
Bloemendaal
Lysbeth Ten
LT
van Wijk
Albert C W A
AC
Galavotti
Daniele
D
van Gulik
Thomas M
TM
Chamuleau
Robert A F M
RA
D000046:Academic Medical Centers; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D049109:Cell Proliferation; D048429:Cell Size; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005786:Gene Expression Regulation / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D008099:Liver / Q000166:cytology* / Q000378:metabolism*; D019164:Liver, Artificial; D009426:Netherlands; D020543:Proteome / Q000378:metabolism*; D013552:Swine; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*
D020543:Proteome