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1.  Discovering Networks of Perturbed Biological Processes in Hepatocyte Cultures 
PLoS ONE  2011;6(1):e15247.
The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs) and collagen sandwiches (CS). Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs). CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.
doi:10.1371/journal.pone.0015247
PMCID: PMC3016309  PMID: 21245926
2.  Metabolic Profiling Based Quantitative Evaluation of Hepatocellular Metabolism in Presence of Adipocyte Derived Extracellular Matrix 
PLoS ONE  2011;6(5):e20137.
The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.
doi:10.1371/journal.pone.0020137
PMCID: PMC3095641  PMID: 21603575
3.  Successful mouse hepatocyte culture with sandwich collagen gel formation 
Purpose
Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking.
Methods
Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied.
Results
After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture.
Conclusion
The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.
doi:10.4174/jkss.2013.84.4.202
PMCID: PMC3616273  PMID: 23577314
Collagen; Culture; Hepatocyte
4.  Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells 
Biomolecules  2013;3(1):242-269.
Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.
doi:10.3390/biom3010242
PMCID: PMC4030890  PMID: 24970167
diazepam; fetal calf serum; poly-HEMA; rat hepatocyte; nonparencymal cells; sandwich model; spheroid model
5.  GENE EXPRESSION PROFILING AND DIFFERENTIATION ASSESSMENT IN PRIMARY HUMAN HEPATOCYTE CULTURES, ESTABLISHED HEPATOMA CELL LINES, AND HUMAN LIVER TISSUES 
Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively overrepresented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in all overrepresented categories, representing such genes as C/EBPα, HNF4α, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined 2-dimensional sandwich system.
doi:10.1016/j.taap.2007.03.032
PMCID: PMC2974173  PMID: 17512962
DNA microarray; human hepatocytes; in vitro hepatic model
6.  Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function 
Hepatology (Baltimore, Md.)  2009;49(2):578-586.
The culture of primary hepatocytes as spheroids creates an efficient 3-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer non-adherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of over 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. In conclusion, spheroid formation by rocker technique was more rapid and more efficient than rotational technique. Rocker formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct.
doi:10.1002/hep.22674
PMCID: PMC2680349  PMID: 19085959
liver tissue construct; bioartificial liver; custom microarray; drug metabolism; spheroid
7.  3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes 
PLoS ONE  2010;5(11):e15456.
Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.
doi:10.1371/journal.pone.0015456
PMCID: PMC2980491  PMID: 21103392
8.  Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human 
PLoS ONE  2009;4(8):e6796.
Background
Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation.
Methodology/Principal Findings
Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8.
Conclusions/Significance
Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.
doi:10.1371/journal.pone.0006796
PMCID: PMC2729378  PMID: 19710929
9.  HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology 
We present HepatoNet1, a manually curated large-scale metabolic network of the human hepatocyte that encompasses >2500 reactions in six intracellular and two extracellular compartments.Using constraint-based modeling techniques, the network has been validated to replicate numerous metabolic functions of hepatocytes corresponding to a reference set of diverse physiological liver functions.Taking the detoxification of ammonia and the formation of bile acids as examples, we show how these liver-specific metabolic objectives can be achieved by the variable interplay of various metabolic pathways under varying conditions of nutrients and oxygen availability.
The liver has a pivotal function in metabolic homeostasis of the human body. Hepatocytes are the principal site of the metabolic conversions that underlie diverse physiological functions of the liver. These functions include provision and homeostasis of carbohydrates, amino acids, lipids and lipoproteins in the systemic blood circulation, biotransformation, plasma protein synthesis and bile formation, to name a few. Accordingly, hepatocyte metabolism integrates a vast array of differentially regulated biochemical activities and is highly responsive to environmental perturbations such as changes in portal blood composition (Dardevet et al, 2006). The complexity of this metabolic network and the numerous physiological functions to be achieved within a highly variable physiological environment necessitate an integrated approach with the aim of understanding liver metabolism at a systems level. To this end, we present HepatoNet1, a stoichiometric network of human hepatocyte metabolism characterized by (i) comprehensive coverage of known biochemical activities of hepatocytes and (ii) due representation of the biochemical and physiological functions of hepatocytes as functional network states. The network comprises 777 metabolites in six intracellular (cytosol, endoplasmic reticulum and Golgi apparatus, lysosome, mitochondria, nucleus, and peroxisome) and two extracellular compartments (bile canaliculus and sinusoidal space) and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. We performed flux-balance analyses to validate whether for >300 different metabolic objectives a non-zero stationary flux distribution could be established in the network. Figure 1 shows one such functional flux mode associated with the synthesis of the bile acid glycochenodeoxycholate, one important hepatocyte-specific physiological liver function. Besides those pathways directly linked to the synthesis of the bile acid, the mevalonate pathway and the de novo synthesis of cholesterol, the flux mode comprises additional pathways such as gluconeogenesis, the pentose phosphate pathway or the ornithine cycle because the calculations were routinely performed on a minimal set of exchangeable metabolites, that is all reactants were forced to be balanced and all exportable intermediates had to be catabolized into non-degradable end products. This example shows how HepatoNet1 under the challenges of limited exchange across the network boundary can reveal numerous cross-links between metabolic pathways traditionally perceived as separate entities. For example, alanine is used as gluconeogenic substrate to form glucose-6-phosphate, which is used in the pentose phosphate pathway to generate NADPH. The glycine moiety for bile acid conjugation is derived from serine. Conversion of ammonia into non-toxic nitrogen compounds is one central homeostatic function of hepatocytes. Using the HepatoNet1 model, we investigated, as another example of a complex metabolic objective dependent on systemic physiological parameters, how the consumption of oxygen, glucose and palmitate is affected when an external nitrogen load is converted in varying proportions to the non-toxic nitrogen compounds: urea, glutamine and alanine. The results reveal strong dependencies between the available level of oxygen and the substrate demand of hepatocytes required for effective ammonia detoxification by the liver.
Oxygen demand is highest if nitrogen is exclusively transformed into urea. At lower fluxes into urea, an intriguing pattern for oxygen demand is predicted: oxygen demand attains a minimum if the nitrogen load is directed to urea, glutamine and alanine with relative fluxes of 0.17, 0.43 and 0.40, respectively (Figure 2A). Oxygen demand in this flux distribution is four times lower than for the maximum (100% urea) and still 77 and 33% lower than using alanine and glutamine as exclusive nitrogen compounds, respectively. This computationally predicted tendency is consistent with the notion that the zonation of ammonia detoxification, that is the preferential conversion of ammonia to urea in periportal hepatocytes and to glutamine in perivenous hepatocytes, is dictated by the availability of oxygen (Gebhardt, 1992; Jungermann and Kietzmann, 2000). The decreased oxygen demand in flux distributions using higher proportions of glutamine or alanine is accompanied by increased uptake of the substrates glucose and palmitate (Figure 2B). This is due to an increased demand of energy and carbon for the amidation and transamination of glutamate and pyruvate to discharge nitrogen in the form of glutamine and alanine, respectively. In terms of both scope and specificity, our model bridges the scale between models constructed specifically to examine distinct metabolic processes of the liver and modeling based on a global representation of human metabolism. The former include models for the interdependence of gluconeogenesis and fatty-acid catabolism (Chalhoub et al, 2007), impairment of glucose production in von Gierke's and Hers' diseases (Beard and Qian, 2005) and other processes (Calik and Akbay, 2000; Stucki and Urbanczik, 2005; Ohno et al, 2008). The hallmark of these models is that each of them focuses on a small number of reactions pertinent to the metabolic function of interest embedded in a customized representation of the principal pathways of central metabolism. HepatoNet1, currently, outperforms liver-specific models computationally predicted (Shlomi et al, 2008) on the basis of global reconstructions of human metabolism (Duarte et al, 2007; Ma and Goryanin, 2008). In contrast to either of the aforementioned modeling scales, HepatoNet1 provides the combination of a system-scale representation of metabolic activities and representation of the cell type-specific physical boundaries and their specific transport capacities. This allows for a highly versatile use of the model for the analysis of various liver-specific physiological functions. Conceptually, from a biological system perspective, this type of model offers a large degree of comprehensiveness, whereas retaining tissue specificity, a fundamental design principle of mammalian metabolism. HepatoNet1 is expected to provide a structural platform for computational studies on liver function. The results presented herein highlight how internal fluxes of hepatocyte metabolism and the interplay with systemic physiological parameters can be analyzed with constraint-based modeling techniques. At the same time, the framework may serve as a scaffold for complementation of kinetic and regulatory properties of enzymes and transporters for analysis of sub-networks with topological or kinetic modeling methods.
We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds.
doi:10.1038/msb.2010.62
PMCID: PMC2964118  PMID: 20823849
computational biology; flux balance; liver; minimal flux
10.  Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection 
PLoS Pathogens  2008;4(8):e1000121.
Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria.
Author Summary
Sporozoites, the infective form of the malaria parasites Plasmodium, are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver where they specifically invade hepatocytes, in which they develop to yield another form, the merozoite, infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. We studied the changes in gene expression in sporozoites, from the parasite species P. falciparum that infects humans, in an in vitro system where they were co-cultured with their natural host cells, primary human hepatocytes. The whole genome transcriptome profiling carried out led to the identification of 532 genes that were up-regulated following co-culture. This genome-wide up-regulation of expression supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes we identified validated our approach as one means to determine the repertoire of proteins implicated during the early events in the infection by P. falciparum, the species responsible for the severest forms of malaria.
doi:10.1371/journal.ppat.1000121
PMCID: PMC2488394  PMID: 18688281
11.  Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase, and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver 
Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P1-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P1-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of 14C-heme in microsomes prepared from hepatocytes derived from rats given 514[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.
PMCID: PMC2595706  PMID: 222079
12.  Engineered Three-Dimensional Liver Mimics Recapitulate Critical Rat-Specific Bile Acid Pathways 
Tissue Engineering. Part A  2010;17(5-6):677-689.
A critical hepatic function is the maintenance of optimal bile acid (BA) compositions to achieve cholesterol homeostasis. BAs are rarely quantified to assess hepatic phenotype in vitro since existing analytical techniques have inadequate resolution. We report a detailed investigation into the biosynthesis and homeostasis of eight primary rat BAs in conventional in vitro hepatocyte cultures and in an engineered liver mimic. The three-dimensional (3D) liver mimic was assembled with layers of primary rat hepatocytes and liver sinusoidal endothelial cells. A high-pressure liquid chromatography and mass spectrometry technique was developed with a detection limit of 1 ng/mL for each BA, which is significantly lower than previous approaches. Over a 2-week culture, only 3D liver mimics exhibited the ratio of conjugated cholic acid to chenodeoxycholic acid that has been observed in vivo. This ratio, an important marker of BA homeostasis, was significantly higher in stable collagen sandwich cultures indicating significant deviation from physiological behavior. The biosynthesis of tauro-β-muricholic acid, a key primary rat BA, doubled only in the engineered liver mimics while decreasing in the other systems. These trends demonstrate that the 3D liver mimics provide a unique platform to study hepatic metabolism.
doi:10.1089/ten.tea.2010.0423
PMCID: PMC3043955  PMID: 20929286
13.  Different Response to Epidermal Growth Factor of Hepatocytes in Cultures Isolated From Male or Female Rat Liver 
Gastroenterology  1987;93(3):597-605.
Deoxyribonucleic acid (DNA) synthesis in hepatocytes isolated from the livers of male and female rats has been compared in monolayer culture. Plating efficiency, DNA and protein content, viability, and morphologic appearance were the same in cultures prepared with hepatocytes isolated from male or female rats. Epidermal growth factor (EGF)-induced DNA synthesis was significantly higher in hepatocytes from male rats than in hepatocytes from female rats. This was the case whether hepatocytes were isolated from normal or partially hepatectomized male or female rats. Hepatocytes isolated from regenerating liver synthesize more DNA than those isolated from normal liver in response to EGF. This increased response to EGF in hepatocytes derived from regenerating liver was relatively the same for male- and female-derived hepatocytes, but the magnitude of the response was considerably higher in male-derived hepatocytes. In contrast, in vivo DNA synthesis in the liver remnant after partial hepatectomy was similar in male and female rats if measured 24 h after the operation. A comparison of EGF binding to male- and female-derived hepatocytes maintained in primary culture indicated a lower number of high-affinity receptors for EGF in the female hepatocytes. The addition of estrogen to primary cultures of hepatocytes isolated from male rats inhibited EGF binding as well as EGF-induced DNA synthesis. Our studies show significant differences in DNA synthesis in response to EGF when male and female hepatocytes are compared in primary culture. The regenerative response after partial hepatectomy, on the other hand, was the same in male and female rats. Thus, our studies indicate that the sex of the donor rat is important when hepatocytes in culture are used for a variety of studies, such as hepatocyte metabolism, induction and control of DNA synthesis, and hepatocarcinogenesis. In addition, our results indicate that caution is advised when inferences are made from in vitro findings for in vivo conditions.
PMCID: PMC2962611  PMID: 3497071
14.  Rat hepatocyte culture model of macrosteatosis: Effect of macrosteatosis induction and reversal on viability and liver-specific function 
Journal of hepatology  2013;59(6):1307-1314.
Background & Aims
A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes.
Methods
Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured.
Results
Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion.
Conclusions
Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.
doi:10.1016/j.jhep.2013.07.019
PMCID: PMC3899833  PMID: 23872604
Steatosis; Triglyceride; Albumin; Urea; Bile; Liver transplantation; Lipid metabolism
15.  Prolonged lidocaine metabolizing activity of primary hepatocytes with spheroid culture using polyurethane foam as a culture substratum 
Cytotechnology  1997;24(3):235-242.
Primary rat hepatocytes formed spheroids in the pores of polyurethane foam (PUF) used as a culture substratum. The hepatocytes in monolayer and spheroid stationary culture converted lidocaine to monoethylglycinexylidide (MEGX) which was N-deethylation of lidocaine. The metabolic activity of the hepatocytes/spheroid stationary culture system was 1.5∼2.0-fold higher than that of monolayer culture for 10 days. The activity of albumin production and cell survival of hepatocytes in monolayer and spheroid cultures decrease due to lidocaine treatment dependend on the lidocaine concentration, but the activity and cell survival in PUF/spheroid stationary culture were maintained at a higher level than that in monolayer culture under the lidocaine treatment. We developed a device for an in vitro liver model, drug metabolism simulator (DMS), using a PUF/spheroid packed-bed module including 4.00 ± 0.68 × 107 hepatocytes and analyzed pharmacokinetics of lidocaine in a one-compartment model. Lidocaine clearance and extraction ratio of hepatocytes in the DMS corresponded to 1.354 ± 0.318 ml/min/g-liver and 0.677 ± 0.0159/g-liver, respectively (N=4). These values were comparable with in vivo values, 1.930 ml/min g-liver and 0.965/g-liver reported by Nyberg (1977). Consequently, PUF/spheroid culture maintained high lidocaine metabolizing activity over a long term and seems to provide a promising culture system as a drug metabolism simulator which will be used for drug screening, cytotoxicity tests and prediction of pharmacokinetics.
doi:10.1023/A:1007935016223
PMCID: PMC3449624  PMID: 22358767
drug metabolism simulator; lidocaine/MEGX; monolayer culture; polyurethane foam; rat hepatocytes; spheroid culture
16.  Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function 
Biomaterials  2012;33(18):4565-4575.
Despite recent advances in biomaterial science, there is yet no culture system that supports long-term culture expansion of human adult hepatocytes, while preserving continued function. Previous studies suggested that acellular liver extracellular matrix (ECM), employed as a substrate, improved proliferation and function of liver cells. Here we investigated whether extracts prepared from acellular liver ECM (liver ECM extract, LEE), or from whole (fresh) liver tissue (liver tissue extract, LTE), could be combined with collagen Type I, hyaluronic acid (HA), or heparin-conjugated HA (HP) hydrogels to enhance survival and functional output of primary human hepatocytes. The liver-specific semi-synthetic ECMs (sECMs) were prepared by incorporating LEE or LTE into the gel matrices. Subsequently, primary human hepatocytes were maintained in sandwich-style hydrogel cultures for 4 weeks. Progressive increase in hepatocyte metabolism was observed in all HA and HP groups. Hepatocytes cultured in HA and HP hydrogels containing LEE or LTE synthesized and secreted steady levels of albumin and urea and sustained cytochrome p450-dependent drug metabolism of ethoxycoumarin. Collectively, these results indicate that customized HA hydrogels with liver-specific ECM components may be an efficient method for expansion human hepatocytes in vitro for cell therapy and drug and toxicology screening purposes.
doi:10.1016/j.biomaterials.2012.03.034
PMCID: PMC3719050  PMID: 22475531
Hyaluronic acid; Heparin; Hepatocyte; Extracellular matrix; Growth factors
17.  A Novel 3D Liver Organoid System for Elucidation of Hepatic Glucose Metabolism 
Biotechnology and bioengineering  2011;109(2):595-604.
Hepatic glucose metabolism is a key player in diseases such as obesity and diabetes as well as in antihyperglycemic drugs screening. Hepatocytes culture in two-dimensional configurations is limited in vitro model for hepatocytes to function properly, while truly practical platforms to perform three-dimensional (3D) culture are unavailable. In this work, we present a practical organoid culture method of hepatocytes for elucidation of glucose metabolism under nominal and stress conditions. Employing this new method of culturing cells within a hollow fiber reactor, hepatocytes were observed to self-assemble into 3D spherical organoids with preservation of tight junctions and display increased liver-specific functions. Compared to both monolayer culture and sandwich culture, the hepatocyte organoids displayed higher intracellular glycogen content, glucose consumption, and gluconeogenesis and approached the in vivo values, as also confirmed by gene expression of key enzymes. Moreover, hepatocyte organoids demonstrated more realistic sensitivity to hormonal challenges with insulin, glucagon, and dexamethasone. Finally, the exposure to high glucose demonstrated toxicities including alteration of mitochondrial membrane potential, lipid accumulation, and reactive oxygen species formation, similar to the in vivo responses, which was not captured by monolayer cultures. Collectively, hepatocyte organoids mimicked the in vivo functions better than hepatocyte monolayer and sandwich cultures, suggesting suitability for applications such as antihyperglycemic drugs screening.
doi:10.1002/bit.23349
PMCID: PMC3907714  PMID: 22006574
rat hepatocytes; 3D organoid culture; hollow fiber bioreactor; glucose metabolism
18.  LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes 
PLoS ONE  2014;9(3):e91921.
Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
doi:10.1371/journal.pone.0091921
PMCID: PMC3958433  PMID: 24643070
19.  System-Driven and Oscillator-Dependent Circadian Transcription in Mice with a Conditionally Active Liver Clock  
PLoS Biology  2007;5(2):e34.
The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs.
Author Summary
In contrast to previously held belief, molecular circadian oscillators are not restricted to specialized pacemaker tissues, such as the brain's suprachiasmatic nucleus (SCN), but exist in virtually all body cells. Although the circadian clocks operative in peripheral cell types are as robust as those residing in SCN neurons, they quickly become desynchronized in vitro due to variations in period length. Hence, in intact animals, the phase coherence between peripheral oscillators must be established by daily signals generated by the SCN master clock. Although the hierarchy between master and slave oscillators is now well established, the respective roles of these clocks in governing the circadian transcription program in a given organ have never been examined. In principle, the circadian expression of genes in a peripheral tissue could be driven either by cyclic systemic cues, by peripheral oscillators, or by both. In order to discriminate between genes regulated by local oscillators and systemic cues in liver, we generated mice in which hepatocyte clocks can be turned on and off at will. These studies suggest that 90% of the circadian transcription program in the liver is abolished or strongly attenuated when hepatocyte clocks are turned off, indicating that the expression of most circadian liver genes is orchestrated by local cellular clocks. The remaining 10% of cyclically expressed liver genes continue to be transcribed in a robustly circadian fashion in the absence of functional hepatocyte oscillators. These genes, which unexpectedly include the bona fide clock gene mPer2, must therefore be regulated by oscillating systemic signals, such as hormones, metabolites, or body temperature. Although temperature rhythms display only modest amplitudes, they appear to play a significant role in the phase entrainment of mPer2 transcription.
Research on mice engineered with an inducible liver clock enabled identification of some genes with expression controlled by the local clock, and other genes (includingmPer2) that maintained circadian oscillations thanks to cues from the SCN.
doi:10.1371/journal.pbio.0050034
PMCID: PMC1783671  PMID: 17298173
20.  The Effectiveness of a Novel Cartridge-Based Bioreactor Design in Supporting Liver Cells 
Tissue Engineering. Part A  2009;15(10):2903-2916.
There are a number of applications—ranging from temporary strategies for organ failure to pharmaceutical testing—that rely on effective bioreactor designs. The significance of these devices is that they provide an environment for maintaining cells in a way that allows them to perform key cellular and tissue functions. In the current study, a novel cartridge-based bioreactor was developed and evaluated. Its unique features include its capacity for cell support and the adaptable design of its cellular space. Specifically, it is able to accommodate functional and reasonably sized tissue (>2.0 × 108 cells), and can be easily modified to support a range of anchorage-dependent cells. To evaluate its efficacy, it was applied to liver support in the current study. This involved evaluating the performance of rat primary hepatocytes within the unique cartridges in culture—sans bioreactor—and after being loaded within the novel bioreactor. Compared to collagen sandwich culture functional controls, hepatocytes within the unique cartridge design demonstrated significantly higher albumin production and urea secretion rates when cultured under dynamic flow conditions—reaching peak values of 170 ± 22 μg/106 cells/day and 195 ± 18 μg/106 cells/day, respectively. The bioreactor's effectiveness in supporting live and functioning primary hepatocytes is also presented. Cell viability at the end of 15 days of culture in the new bioreactor was 84 ± 18%, suggesting that the new design is effective in maintaining primary hepatocytes for at least 2 weeks in culture. Liver-specific functions of urea secretion, albumin synthesis, and cytochrome P450 activity were also assessed. The results indicate that hepatocytes are able to achieve good functional performance when cultured within the novel bioreactor. This is especially true in the case of cytochrome P450 activity, where by day 15 of culture, hepatocytes within the bioreactor reached values that were 56.6% higher than achieved by the collagen sandwich functional control cultures. The success of the novel cartridge-based bioreactor in supporting hepatocytes with good viability and functional performance suggests that it is an effective design for supporting anchorage-dependent cells.
doi:10.1089/ten.tea.2008.0279
PMCID: PMC2792046  PMID: 19271993
21.  Influence of Seeding Density and Extracellular Matrix on Bile Acid Transport and Mrp4 Expression in Sandwich-Cultured Mouse Hepatocytes 
Molecular pharmaceutics  2010;7(2):491-500.
This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat™ plates with an overlay of Matigel™ (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25 or 1.5 × 106 cells/well. The lower seeding densities of 1.0 and 1.25 × 106 cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5- (and 6)-carboxy-2′,7′dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased ~2- to 3-fold between seeding densities of 1.0 × 106 and 1.25 × 106 cells/well regardless of extracellular matrix; an additional ~3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 × 106 and 1.5 × 106 cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 × 106 cells/well or below, and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes.
doi:10.1021/mp900227a
PMCID: PMC3235796  PMID: 19968322
Sandwich-cultured mouse hepatocytes; Mrp4; taurocholate; BEI; in vitro Clbiliary; carboxydichlorofluorescein diacetate
22.  Peroxisome Proliferator-Activated Receptor α Activates Human Multidrug Resistance Transporter 3/ATP-Binding Cassette Protein Subfamily B4 Transcription and Increases Rat Biliary Phosphatidylcholine Secretion 
Hepatology (Baltimore, Md.)  2014;59(3):1030-1042.
Multidrug resistance transporter 3/ATP-binding cassette protein subfamily B4 (MDR3/ABCB4) is a critical determinant of biliary phosphatidylcholine (PC) secretion. Clinically, mutations and partial deficiencies in MDR3 result in cholestatic liver injury. Thus, MDR3 is a potential therapeutic target for cholestatic liver disease. Fenofibrate is a peroxisome proliferator-activated receptor (PPAR) α ligand that has antiinflammatory actions and regulates bile acid detoxification. Here we examined the mechanism by which fenofibrate regulates MDR3 gene expression. Fenofibrate significantly up-regulated MDR3 messenger RNA (mRNA) and protein expression in primary cultured human hepatocytes, and stimulated MDR3 promoter activity in HepG2 cells. In silico analysis of 5′-upstream region of human MDR3 gene revealed a number of PPARα response elements (PPRE). Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated specific binding of PPARα to the human MDR3 promoter. Targeted mutagenesis of three novel PPREs reduced inducibility of the MDR3 promoter by fenofibrate. In collagen sandwich cultured rat hepatocytes, treatment with fenofibrate increased secretion of fluorescent PC into bile canaliculi.
Conclusion
Fenofibrate transactivates MDR3 gene transcription by way of the binding of PPARα to three novel and functionally critical PPREs in the MDR3 promoter. Fenofibrate treatment further stimulates biliary phosphatidylcholine secretion in rat hepatocytes, thereby providing a functional correlate. We have established a molecular mechanism that may contribute to the beneficial use of fenofibrate therapy in human cholestatic liver disease.
doi:10.1002/hep.26894
PMCID: PMC4049334  PMID: 24122873
23.  Development of a bioartificial liver employing xenogeneic hepatocytes 
Cytotechnology  1997;23(1-3):29-38.
Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations.
doi:10.1023/A:1007906512616
PMCID: PMC3449881  PMID: 22358518
24.  Characterization of the Secreted Proteome of Rat Hepatocytes Cultured in Collagen Sandwiches 
Chemical research in toxicology  2005;18(7):1132-1139.
Analysis of proteins in biological samples opens up the possibility of discovering new markers of toxicity. The liver is one of the primary targets of drug-induced toxicity and it also secretes many plasma proteins, which can be measured clinically. Most of the plasma proteins secreted by the liver are secreted by hepatocytes, but there is little information regarding the protein profile secreted by these cells. The purpose of this study was to analyze the secreted proteome of primary rat hepatocytes in a collagen gel sandwich configuration by a Gel-LC-MS/MS procedure. We identified over 600 peptides corresponding to more than 200 proteins. The protein profile included over 50 plasma proteins secreted by the liver, suggesting that the cultured hepatocytes secrete many of the proteins that they produce in vivo. Our data also suggests that the hepatocytes are actively remodeling their environment, since we identified several structural extracellular matrix proteins as well as some proteins known to be secreted specifically during liver regeneration. We also identified two proteins, α1-antitrypsin and α2-macroglobulin, whose secretions appear to be down-regulated in cells exposed to aflatoxin B1. It was noted that a 15 nM dose of aflatoxin B1 led to substantially diminished levels of these proteins and that day 6 of incubation was the ideal timepoint for medium collection. These data suggest that proteins in the conditioned medium of hepatocyte sandwich culture might lead to discovery of biomarkers for drug chemical toxicity.
doi:10.1021/tx0500225
PMCID: PMC4113969  PMID: 16022505
hepatocytes; sandwich; proteome; proteomics; aflatoxin
25.  Layered Long Term Co-Culture of Hepatocytes and Endothelial Cells on a Transwell Membrane: Toward Engineering the Liver Sinusoid 
Biofabrication  2013;5(4):045008.
This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin matrigel layer placed between hepatocytes and endothelial cells to mimic the Space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the co-cultured system from Day 13 to Day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells.
doi:10.1088/1758-5082/5/4/045008
PMCID: PMC3935322  PMID: 24280542
Liver model; Liver co-culture; Primary Rat Hepatocytes; Liver sinusoidal endothelial cell; Transwell

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