Leonard J. Nelson

The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes

The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 10(8) porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P < 0.01), and significantly smaller pyruvate (day 1 vs day 14 P < 0.03) and threonine consumption (day 1 vs day 10 P < 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P < 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P < 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.

Low-Shear Modelled Microgravity Environment Maintains Morphology and Differentiated Functionality of Primary Porcine Hepatocyte Cultures

Hepatocytes cultured in conventional static culture rapidly lose polarity and differentiated function. This could be explained by gravity-induced sedimentation, which prevents formation of complete three-dimensional (3D) cell-cell/cell-matrix interactions and disrupts integrin-mediated signals (including the most abundant hepatic integrin α5β1), important for cellular polarity and differentiation. Cell culture in a low fluid shear modelled microgravity (about 10–2g) environment promotes spatial colocation/self-aggregation of dissociated cells and induction of 3D differentiated liver morphology. Previously, we demonstrated the utility of a NASA rotary bioreactor in maintaining key metabolic functions and 3D aggregate formation of high-density primary porcine hepatocyte cultures over 21 days. Using serum-free chemically defined medium, without confounding interactions of exogenous bioscaffolding or bioenhancing surface materials, we investigated features of hepatic cellular polarity and differentiated functionality, including expression of hepatic integrin α5, as markers of functional morphology. We report here that in the absence of exogenous biomatrix scaffolding, hepatocytes cultured in serum-free chemically defined medium in a microgravity environment rapidly (<24 h) form macroscopic (2–5 mm), compacted 3D hepatospheroid structures consisting of a shell of glycogen-positive viable cells circumscribing a core of eosinophilic cells. The spheroid shell layers exhibited ultrastructural, morphological and functional features of differentiated, polarized hepatic tissue including strong expression of the integrin α5 subunit, functional bile canaliculi, albumin synthesis, and fine ultrastructure reminiscent of in vivo hepatic tissue. The low fluid shear microgravity environment may promote tissue-like self-organization of dissociated cells, and offer advantages over spheroids cultured in conventional formats to delineate optimal conditions for enhanced directed tissue self-assembly.

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure

BackgroundThe development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF.Method35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased.ResultsCytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 μmol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischers ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06.ConclusionWe have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model

Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity, and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis, and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells.

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs) - integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications.

Human hepatic HepaRG cells maintain an organotypic phenotype with high intrinsic CYP450 activity/metabolism and significantly outperform standard HepG2/C3A cells for pharmaceutical and therapeutic applications

Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver‐specific functions such as CYP450 activity. Alternatives include the HepG2‐derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co‐culture model of hepatocytes and cholangiocytes reported to maintain in vivo‐like liver‐specific functions, including intact Phase I–III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre‐clinical drug testing or therapeutics. Compared with C3As, HepaRG co‐cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5β1 – an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC‐MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I–II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.

Fibrinogen production is enhanced in an in-vitro model of non-alcoholic fatty liver disease: An isolated risk factor for cardiovascular events?

BackgroundCardiovascular disease (CVD) remains the major cause of excess mortality in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the individual contribution of NAFLD to CVD risk factors in the absence of pathogenic influences from other comorbidities often found in NAFLD patients, by using an established in-vitro model of hepatic steatosis.MethodsHistopathological events in non-alcoholic fatty liver disease were recapitulated by focused metabolic nutrient overload of hepatoblastoma C3A cells, using oleate-treated-cells and untreated controls for comparison. Microarray and proteomic data from cell culture experiments were integrated into a custom-built systems biology database and proteogenomics analysis performed. Candidate genes with significant dysregulation and concomitant changes in protein abundance were identified and STRING association and enrichment analysis performed to identify putative pathogenic pathways.ResultsThe search strategy yielded 3 candidate genes that were specifically and significantly up-regulated in nutrient-overloaded cells compared to untreated controls: fibrinogen alpha chain (2.2 fold), fibrinogen beta chain (2.3 fold) and fibrinogen gamma chain (2.1 fold) (all rank products pfp <0.05). Fibrinogen alpha and gamma chain also demonstrated significant concomitant increases in protein abundance (3.8-fold and 2.0-fold, respectively, p <0.05).ConclusionsIn-vitro modelling of NAFLD and reactive oxygen species formation in nutrient overloaded C3A cells, in the absence of pathogenic influences from other comorbidities, suggests that NAFLD is an isolated determinant of CVD. Nutrient overload-induced up-regulation of all three fibrinogen component subunits of the coagulation cascade provides a possible mechanism to explain the excess CVD mortality observed in NAFLD patients.

Comparison of Bioenergetic Activity of Primary Porcine Hepatocytes Cultured in Four Different Media

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were Williams E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 × 1010; viability 87 ± 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 × 106 viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis, ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. Williams E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.

Proteomic profiling of cellular steatosis with concomitant oxidative stress in vitro.

BackgroundNutrient excess underpins the development of nonalcoholic fatty liver disease (NAFLD). The ensuing metabolic derangement is characterised by increased cellular respiration, oxidative stress and mitochondrial impairment. We have previously recapitulated these events in an in vitro cellular steatosis model. Here, we examined the distinct patterns of protein expression involved using a proteomics approach.MethodsHuman hepatoblastoma C3A cells were treated with a combination of energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N). Proteins extracts were trypsinized and analyzed on a capillary HPLC OrbitrapXL mass spectrometer. Proteins were quantified using a label-free intensity based approach. Functional enrichment analysis was performed using ToppCluster via Gene Ontology (GO) database.ResultsOf the 1327 proteins identified, 104 were differentially expressed between LPON and untreated cells (defined as: ≥2 peptides; fold change ≥1.5; p-value <0.05). Seventy of these were upregulated with LPON. Functional enrichment analysis revealed enhanced protein biosynthesis accompanied by downregulation of histones H2A type 1-A, H1.2, H1.5 and H1.0I in LPON cells. Lipid binding annotations were also enriched as well as proteins involved in cholesterol synthesis, uptake and efflux. Increased expression of aldo-keto reductase family 1, member C1 and C3 suggests enhanced sterol metabolism and increased ROS-mediated lipid peroxidation.ConclusionsThe surge of energy substrates diverts free fatty acid metabolism towards pathways that can mitigate lipotoxicity. The histones depletion may represent an adaptation to increased protein synthesis. However, this can also expose DNA to oxidative stress thus should be explored further in the context of NAFLD progression.

Krüppel-like factor 6 is a transcriptional activator of autophagy in acute liver injury

Krüppel-like factor 6 (KLF6) is a transcription factor and tumor suppressor. We previously identified KLF6 as mediator of hepatocyte glucose and lipid homeostasis. The loss or reduction of KLF6 is linked to the progression of hepatocellular carcinoma, but its contribution to liver regeneration and repair in acute liver injury are lacking so far. Here we explore the role of KLF6 in acute liver injury models in mice, and in patients with acute liver failure (ALF). KLF6 was induced in hepatocytes in ALF, and in both acetaminophen (APAP)- and carbon tetrachloride (CCl4)-treated mice. In mice with hepatocyte-specific Klf6 knockout (DeltaKlf6), cell proliferation following partial hepatectomy (PHx) was increased compared to controls. Interestingly, key autophagic markers and mediators LC3-II, Atg7 and Beclin1 were reduced in DeltaKlf6 mice livers. Using luciferase assay and ChIP, KLF6 was established as a direct transcriptional activator of ATG7 and BECLIN1, but was dependent on the presence of p53. Here we show, that KLF6 expression is induced in ALF and in the regenerating liver, where it activates autophagy by transcriptional induction of ATG7 and BECLIN1 in a p53-dependent manner. These findings couple the activity of an important growth inhibitor in liver to the induction of autophagy in hepatocytes.