HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

Cell Physiol Biochem. 2018;48(5):2189-2204. doi: 10.1159/000492560. Epub 2018 Aug 15.


Background/aims: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective.

Methods: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription.

Results: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible.

Conclusion: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.

Keywords: 3D culture; Bioartifcial liver; Dedifferentiation; HepaRG; Liver failure; Plasma toxicity.

MeSH terms

  • Animals
  • Arginase / genetics
  • Arginase / metabolism
  • Cell Culture Techniques / methods*
  • Cell Differentiation / drug effects
  • Culture Media / pharmacology
  • Cytochrome P-450 CYP3A / genetics
  • Cytochrome P-450 CYP3A / metabolism
  • Female
  • Hepatocytes / cytology
  • Hepatocytes / metabolism
  • Humans
  • Liver Failure, Acute / metabolism
  • Liver Failure, Acute / pathology*
  • Liver, Artificial*
  • Male
  • Membrane Potential, Mitochondrial / drug effects
  • Plasma / chemistry*
  • Plasma / metabolism
  • Stem Cells / cytology
  • Stem Cells / metabolism
  • Swine
  • Up-Regulation / drug effects


  • Culture Media
  • Cytochrome P-450 CYP3A
  • Arginase