Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells.

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.

End-stage liver failure: filling the treatment gap at the intensive care unit.

End-stage liver failure is a condition of collapsing liver function with mortality rates up to 80. Liver transplantation is the only lifesaving therapy. There is an unmet need for therapy to extend the waiting time for liver transplantation or regeneration of the native liver. Here we review the state-of-the-art of non-cell based and cell-based artificial liver support systems, cell transplantation and plasma exchange, with the first therapy relying on detoxification, while the others aim to correct also other failing liver functions and/or modulate the immune response. Meta-analyses on the effect of non-cell based systems show contradictory outcomes for different types of albumin purification devices. For bioartificial livers proof of concept has been shown in animals with liver failure. However, large clinical trials with two different systems did not show a survival benefit. Two clinical trials with plasma exchange and one with transplantation of mesenchymal stem cells showed positive outcomes on survival. Detoxification therapies lack adequacy for most patients. Correction of additional liver functions, and also modulation of the immune system hold promise for future therapy of liver failure.

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

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.

Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.

Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.

Phase 1 and phase 2 drug metabolism and bile acid production of HepaRG cells in a bioartificial liver in absence of dimethyl sulfoxide.

The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.

A systematic review on prognostic indicators of acute on chronic liver failure and their predictive value for mortality.

BACKGROUND: An early and proper diagnosis of acute on chronic liver failure (ACLF), together with the identification of indicators associated with disease severity is critical for outcome prediction and therapy. OBJECTIVE: To systematically identify and summarize prognostic indicators for patients with ACLF and to evaluate the predictive value of these indicators. METHODS: Embase and Ovid-Medline were searched for English-language articles. The search criteria focused on identifying clinical trials and observational studies reporting on indicators used for prediction of mortality in patients with ACLF. RESULTS: Of 2382 studies identified, 19 were included for detailed analysis. Thirteen different definitions of ACLF were found. The main differences were related to acute deterioration in liver function, coagulopathy and hyperbilirubinaemia/jaundice. Seventy three prognostic indicators and their association with mortality were extracted and categorized into seven categories: general markers (n = 13), viral markers (n = 6), bio-markers (n = 22), hemodynamics (n = 1), morphology/histology (n = 17), scoring systems (n = 10) and treatments (n = 4). CONCLUSIONS: The ambiguity and variability in the definition of ACLF and in its predictive indicators hampers comparability among studies. There is a need for a single uniform definition of ACLF. Also absence of a gold standard is an obstacle to render one indicator superior to another. The age, hepatic encephalopathy, model for end-stage liver disease score, total bilirubin and International normalized ratio (prothrombin time) appeared to be promising candidates for evaluation in future studies. The result of this review may be useful as a starting point in developing a standard list of indicators for clinical outcome that concur with the clinicians' subjective views on prognosis in ACLF.

Effects of acute-liver-failure-plasma exposure on hepatic functionality of HepaRG-AMC-bioartificial liver.

BACKGROUND & AIMS: The AMC-bioartificial liver loaded with the human hepatoma cell line HepaRG as biocomponent (HepaRG-AMC-BAL) has recently proven efficacious in rats with acute liver failure (ALF). However, its efficacy may be affected by cytotoxic components of ALF plasma during treatment. In this study, we investigated the effects of ALF-plasma on the HepaRG-AMC-BAL. METHODS: HepaRG-AMC-BALs were connected to the blood circulation of rats with total liver ischaemia, either during the first 5 h after induction of ischaemia (mild ALF group), or during the following 10 h (severe ALF group). After disconnection, the BALs were assessed for cell leakage, gene transcript levels, ammonia elimination, urea production, cytochrome P450 3A4 activity, apolipoprotein A 1 production, glucose and amino acid metabolism. RESULTS: Cell leakage increased 2.5-fold in the severe ALF group, but remained limited in all groups. Hepatic gene transcript levels decreased (max 40-fold) or remained stable. In contrast, hepatic functions increased slightly or remained stable. Particularly, urea production increased 1.5-fold, with a concurrent increase in arginase 2 transcription and arginine consumption, with a trend towards reduced conversion of ammonia into urea. The amino acid consumption increased, however, the net glucose consumption remained stable. CONCLUSIONS: The HepaRG-AMC-BAL retains functionality after both mild and severe exposure to ALF plasma, but urea production may be increasingly derived from arginase 2 activity instead of urea cycle activity. Nevertheless, the increase in cell leakage and decrease in various hepatic transcript levels suggest that a decrease in hepatic functionality may follow upon extended exposure to ALF plasma.

Perfusion flow rate substantially contributes to the performance of the HepaRG-AMC-bioartificial liver.

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver-failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC-BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6ß-hydroxylation of testosterone showed 2-20-fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG-BAL); and (3) in the current set-up the mass balance of substrate, metabolite, or cell damage markers between in-and out-flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG-BAL).

A comparison of RIFLE with and without urine output criteria for acute kidney injury in critically ill patients.

INTRODUCTION: The Risk, Injury, Failure, Loss, and End-Stage Renal Disease (RIFLE) is a consensus-based classification system for diagnosing acute kidney insufficiency (AKI), based on serum creatinine (SCr) and urine output criteria (RIFLESCr+UO). The urine output criteria, however, are frequently discarded and many studies in the literature applied only the SCr criteria (RIFLESCr). We diagnosed AKI using both RIFLE methods and compared the effects on time to AKI diagnosis, AKI incidence and AKI severity. METHODS: This was a prospective observational cohort study during four months in adult critically ill patients admitted to the ICU for at least 48 hours. During the first week patients were scored daily for AKI according to RIFLESCr+UO and RIFLESCr. We assessed urine output hourly and fluid balance daily. The baseline SCr was estimated if a recent pre-ICU admission SCr was unknown. Based on the two RIFLE methods for each patient we determined time to AKI diagnosis (AKI-0) and maximum RIFLE grade. RESULTS: We studied 260 patients. A pre-ICU admission SCr was available in 101 (39%) patients. The two RIFLE methods resulted in statistically significantly different outcomes for incidence of AKI, diagnosis of AKI for individual patients, distribution of AKI-0 and distribution of the maximum RIFLE grade. Discarding the RIFLE urine criteria for AKI diagnosis significantly underestimated the presence and grade of AKI on admission and during the first ICU week (P < 0,001) and significantly delayed the diagnosis of AKI (P < 0.001). Based on RIFLESCr 45 patients had no AKI on admission but subsequently developed AKI. In 24 of these patients (53%) AKI would have been diagnosed at least one day earlier if the RIFLE urine criteria had been applied. Mortality rate in the AKI population was 38% based on RIFLESCr and 24% based on RIFLESCr+UO (P = 0.02). CONCLUSIONS: The use of RIFLE without the urine criteria significantly underscores the incidence and grade of AKI, significantly delays the diagnosis of AKI and is associated with higher mortality.

Stable overexpression of pregnane X receptor in HepG2 cells increases its potential for bioartificial liver application.

To bridge patients with acute liver failure to transplantation or liver regeneration, a bioartificial liver (BAL) is urgently needed. A BAL consists of an extracorporeal bioreactor loaded with a bioactive mass that would preferably be of human origin and display high hepatic functionality, including detoxification. The human hepatoma cell line HepG2 exhibits many hepatic functions, but its detoxification function is low. In this study, we investigated whether stable overexpression of pregnane X receptor (PXR), a master regulator of diverse detoxification functions in the liver [eg, cytochrome P450 3A (CYP3A) activity], would increase the potential of HepG2 for BAL application. Stable overexpression was achieved by lentiviral expression of the human PXR gene, which yielded cell line cBAL119. In monolayer cultures of cBAL119 cells, PXR transcript levels increased 29-fold versus HepG2 cells. Upon activation of PXR by rifampicin, the messenger RNA levels of CYP3A4, CYP3A5, and CYP3A7 increased 49- to 213-fold versus HepG2 cells. According to reporter gene assays with different inducers, the highest increase in CYP3A4 promoter activity (131-fold) was observed upon induction with rifampicin. Inside BALs, the proliferation rates, as measured by the DNA content, were comparable between the 2 cell lines. The rate of testosterone 6beta-hydroxylation, a measure of CYP3A function inside BALs, increased 4-fold in cBAL119 BALs versus HepG2 BALs. Other functions, such as apolipoprotein A1 synthesis, urea synthesis, glucose consumption, and lactate production, remained unchanged or increased. Thus, stable PXR overexpression markedly increases the potential of HepG2 for BAL application.

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver.

BACKGROUND: Clinical use of porcine cell-based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow-up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center-BAL (AMC-BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real-time polymerase chain reaction assay has been used. RESULTS: Eight patients who received a liver transplant after AMC-BAL treatment are still alive under long-term pharmacological immunosuppression. The current clinical follow-up ranges from 5.6 to 8.7 yr after BAL treatment. A new q-real-time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥ 5 copies per 1 × 10(5) PBMCs. The linear dynamic range was from 5 × 10(0) to 5 × 10(6) copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. CONCLUSION: Up to 8.7 yr after exposure to treatment with porcine liver cell-based BAL, no PERV infection has been found in long-term immunosuppressed patients and in HCWs by a new highly sensitive and specific q-real-time PCR assay.

Genome-wide expression profiling reveals increased stability and mitochondrial energy metabolism of the human liver cell line HepaRG-CAR.

Human liver cell line HepaRG is a well-known source of human hepatocyte-like cells which, however, displays limited biotransformation and a tendency to transform after 20 passages. The new HepaRG-CAR cell line overexpressing constitutive androstane receptor (CAR, NR1I3), a regulator of detoxification and energy metabolism outperforms the parental HepaRG cell line in various liver functions. To further characterize this cell line and assess its stability we compared HepaRG-CAR with HepaRG cells at different passages for their expression profile, ammonia and lactate metabolism, bile acid and reactive oxygen species (ROS) production. Transcriptomic profiling of HepaRG-CAR vs. HepaRG early-passage revealed downregulation of hypoxia, glycolysis and proliferation and upregulation of oxidative phosphorylation genesets. In addition CAR overexpression downregulated the mTORC1 signaling pathway, which, as mediator of proliferation and metabolic reprogramming, may play an important role in the establishment of the HepaRG-CAR phenotype. The ammonia and lactate metabolism and bile acid production of HepaRG-CAR cells was stable for 10 additional passages compared to HepaRG cells. Interestingly, bile acid production was 4.5-fold higher in HepaRG-CAR vs. HepaRG cells, whereas lactate and ROS production were 2.7- and 2.0-fold lower, respectively. Principal component analysis showed clustering of HepaRG-CAR (early- and late-passage) and HepaRG early-passage and not with HepaRG late-passage indicating that passaging exerted larger effect on the transcriptional profile of HepaRG than HepaRG-CAR cells. In conclusion, overexpression of CAR in HepaRG cells improves their bile acid production, mitochondrial energy metabolism, and stability, with the latter possibly due to reduced ROS production, resulting in an optimized source of human hepatocytes.

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

BACKGROUND: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL). METHODS: Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well. RESULTS: Viable hepatocyte layers of approximately 150 microm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis. CONCLUSION: Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4alpha transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the beta-catenin signaling pathway.

Enhanced oxygen availability improves liver-specific functions of the AMC bioartificial liver.

Long-term culturing of primary porcine hepatocytes (PPH) inside the Academic Medical Center (AMC)-bioartificial liver is characterized by increased anaerobic glycolysis. Recommendations to increase oxygen availability were proposed in a previous numerical study and were experimentally evaluated in this study. Original bioreactors as well as new configuration bioreactors with 2.2-fold thinner nonwoven matrix and 2-fold more capillaries were loaded with PPHs and oxygenated with different gas oxygen pressures resulting in medium pO(2) (pO(2-med)) of either 135-150 mm Hg or 235-250 mm Hg. After 6 days culturing, new configuration bioreactors with pO(2-med )of 250 mm Hg showed significantly reduced anaerobic glycolysis, 60% higher liver-specific functions, and increased transcript levels of five liver-specific genes compared to the standard bioreactor cultures. Changed bioreactor configuration and increasing pO(2-med) contributed equally to these improvements. Histological examination demonstrated small differences in cell organization. In conclusion, higher metabolic stability and liver-specific functionality was achieved by enhanced oxygen availability based on a prior modeling concept.

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines.

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.

AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration.

BACKGROUND: Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we investigated the background of the differences between BAL-cultures and monolayers. METHODS: We performed whole-genome microarray analysis on HepaRG monolayer and BAL-cultures. Next, mitochondrial biogenesis was studied in monolayer and BAL-cultures of HepaRG and C3A. The driving forces for mitochondrial biogenesis by BAL-culturing were investigated in representative culture models differing in oxygenation level, medium flow or 2D vs 3D configuration. RESULTS: Gene-sets related to mitochondrial energy metabolism were most prominently up-regulated in HepaRG-BAL vs monolayer cultures. This was confirmed by a 2.4-fold higher mitochondrial abundance with increased expression of mitochondrial OxPhos complexes. Moreover, the transcript levels of mitochondria-encoded genes were up to 3.6-fold induced and mitochondrial membrane potential activity was 8.3-fold increased in BAL vs monolayers. Culturing with 40% O2, dynamic medium flow and/or in 3D increased the mitochondrial abundance and expression of mitochondrial complexes vs standard monolayer culturing. The stimulatory effect of the BAL culture on mitochondrial biogenesis was confirmed in C3A cells in which mitochondrial abundance increased 2.2-fold with induction of mitochondria-encoded genes. CONCLUSIONS AND GENERAL SIGNIFICANCE: The increased functionality of liver cell lines upon AMC-BAL culturing is associated with increased mitochondrial biogenesis. High oxygenation, medium perfusion and 3D configuration contribute to the up-regulation of the mitochondrial biogenesis.

Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.