The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information and their shortcomings have resulted in ‘silent’ hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells and endothelial cells; and, b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.
Drug Induced Liver Injury; Liver on chip; Hepatotoxicity; High Content Screening; Predictive Modeling
Skin sensitization remains a major environmental and occupational health hazard. Animal models have been used as the gold standard method of choice for estimating chemical sensitization potential. However, a growing international drive and consensus for minimizing animal usage have prompted the development of in vitro methods to assess chemical sensitivity. In this paper, we examine existing approaches including in silico models, cell and tissue based assays for distinguishing between sensitizers and irritants. The in silico approaches that have been discussed include Quantitative Structure Activity Relationships (QSAR) and QSAR based expert models that correlate chemical molecular structure with biological activity and mechanism based read-across models that incorporate compound electrophilicity. The cell and tissue based assays rely on an assortment of mono and co-culture cell systems in conjunction with 3D skin models. Given the complexity of allergen induced immune responses, and the limited ability of existing systems to capture the entire gamut of cellular and molecular events associated with these responses, we also introduce a microfabricated platform that can capture all the key steps involved in allergic contact sensitivity. Finally, we describe the development of an integrated testing strategy comprised of two or three tier systems for evaluating sensitization potential of chemicals.
Immunobiology; Skin sensitization; In silico approaches; QSAR; 2D cell based models; 3D skin tissue models; Integrated Testing Strategies; Microfabrication; Microfluidics
Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.
Non-viral transfection; Lentiviruses; Primary rat hepatocytes; Huh7.5.1; Transfection efficiency; Hepatic function
The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.
Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.
Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability.
Rat livers were cold-stored for 0, 24, 48, 72, and 120 hours, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 hours were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP.
ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 hours of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 hours of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 hours.
Vascular resistance and ATP recovery suggest a decrease in viability after 48 hours of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 hours of SCS.
Liver; Transplantation; Machine Perfusion; Preservation; Cold Ischemia; Adenosine Triphosphate
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.
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.
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.
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.
Steatosis; Triglyceride; Albumin; Urea; Bile; Liver transplantation; Lipid metabolism
The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3 hour room temperature (20±2°C) machine perfusion protocol to treat non-premedicated livers exposed to 1 hour of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers, and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Further, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized.
DCD; NHBD; ischemia; ATP; cell isolation
Purpose of review
The capacity of the liver to regenerate and maintain a constant size despite injury is unique. However, the exact mechanisms are not completely clear. Cell transplantation has been proposed as an alternative treatment of liver diseases. Recent progress has been reported on the generation of stem/progenitor cells that may differentiate towards the hepatic lineage. However, it is currently difficult to determine which of the stem/progenitor cell populations are the best for therapy of a given disease.
The limited access to donor human hepatocytes has opened a great interest on the generation of hepatocyte-like cells. Several potential cell sources have been identified. However, general standardization of the methods to evaluate these cells is particularly important for the promise of stem/progenitor-derived hepatocyte-based therapies. Moreover, innovations aimed at improving hepatocyte delivery, survival and engraftment have recently opened the field of organ engineering that may improve the perspective of liver repopulation.
Here we review current evidence reported from the perspective of potential clinical applications of different hepatic cell sources with repopulation capacities and the future perspectives and tools that can facilitate the translation of laboratory work into clinical success.
Hepatocyte transplantation; xenogenic hepatocytes; stem cell-derived hepatocytes; liver tissue engineering
Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.
Cytokine; Immune response; Immunomodulation; Macrophage; Microencapsulation; Vaccine
Orthotopic liver transplantation is the only definitive treatment for end stage liver failure and the shortage of donor organs severely limits the number of patients receiving transplants. Liver tissue engineering aims to address the donor liver shortage by creating functional tissue constructs to replace a damaged or failing liver. Despite decades of work, various bottoms-up, synthetic biomaterials approaches have failed to produce a functional construct suitable for transplantation. Recently, a new strategy has emerged using whole organ scaffolds as a vehicle for tissue engineering. This technique involves preparation of these organ scaffolds via perfusion decellularization with the resulting scaffold retaining the circulatory network of the native organ. This important phenomenon allows for the construct to be repopulated with cells and to be connected to the blood torrent upon transplantation. This opinion paper presents the current advances and discusses the challenges of creating fully functional transplantable liver grafts with this whole liver engineering approach.
Bulge stem cells reside in the lowest permanent portion of hair follicles and are responsible for the renewal of these follicles along with the repair of the epidermis during wound healing. These cells are identified by surface expression of CD34 and the α6-integrin. When CD34 and α6 double-positive cells are isolated and implanted into murine skin, they give rise to epidermis and hair follicle structures. The current gold standard for isolation of these stem cells is fluorescence-activated cell sorting (FACS) based on cell surface markers. Here, we describe an alternative method for CD34 bulge stem cell isolation: a microfluidic platform that captures stem cells based on cell surface markers. This method is relatively fast, requiring 30 min of time from direct introduction of murine skin tissue digestate into a two-stage microfluidic device to one-pass elution of CD34+ enriched cells with a purity of 55.8%±5.1%. The recovered cells remain viable and formed colonies with characteristic morphologies. When grown in culture, enriched cells contain a larger α6+ population than un-enriched cells.
Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.
Diffuse axonal injury (DAI) is a devastating consequence of traumatic brain injury, resulting in significant axon and neuronal degeneration. Currently, therapeutic options are limited. Using our brain-on-a-chip device, we evaluated axonal responses to DAI. We observed that axonal diameter plays a significant role in response to strain injury, which correlated to delayed elasticity and inversely correlated to axonal beading and axonal degeneration. When changes in mitochondrial membrane potential (MMP) were monitored an applied strain injury threshold was noted, below which delayed hyperpolarization was observed and above which immediate depolarization occurred. When the NHE-1 inhibitor EIPA was administered before injury, inhibition in both hyperpolarization and depolarization occurred along with axonal degeneration. Therefore, axonal diameter plays a significant role in strain injury and our brain-on-a-chip technology can be used both to understand the biochemical consequences of DAI and screen for potential therapeutic agents.
Emerging bacterial resistance to multiple drugs is an increasing problem in burn wound management. New non-pharmacologic interventions are needed for burn wound disinfection. Here we report on a novel physical method for disinfection: antiseptic pulsed electric field (PEF) applied externally to the infected burns. In a mice model, we show that PEF can reduce the load of multidrug resistant Acinetobacter baumannii present in a full thickness burn wound by more than four orders of magnitude, as detected by bioluminescence imaging. Furthermore, using a finite element numerical model, we demonstrate that PEF provides non-thermal, homogeneous, full thickness treatment for the burn wound, thus, overcoming the limitation of treatment depth for many topical antimicrobials. These modeling tools and our in vivo results will be extremely useful for further translation of the PEF technology to the clinical setting, as they provide the essential elements for planning of electrode design and treatment protocol.
The resolution of type 2 diabetes after Roux-en-Y gastric bypass (RYGB) attests to the important role of the gastrointestinal tract in glucose homeostasis. Previous studies in RYGB-treated rats have shown that the Roux limb displays hyperplasia and hypertrophy. Here, we report that the Roux limb of RYGB-treated rats exhibits reprogramming of intestinal glucose metabolism to meet its increased bioenergetic demands; glucose transporter-1 is up-regulated, basolateral glucose uptake is enhanced, aerobic glycolysis is augmented, and glucose is directed toward metabolic pathways that support tissue growth. We show that reprogramming of intestinal glucose metabolism is triggered by the exposure of the Roux limb to undigested nutrients. We demonstrate by positron emission tomography–computed tomography scanning and biodistribution analysis using 2-deoxy-2-[18F]fluoro-D-glucose that reprogramming of intestinal glucose metabolism renders the intestine a major tissue for glucose disposal, contributing to the improvement in glycemic control after RYGB.
Organ culture systems are instrumental as experimental whole-organ models of physiology and disease, as well as preservation modalities facilitating organ replacement therapies such as transplantation. Nevertheless, a coordinated system of machine perfusion components and integrated regulatory control has yet to be fully developed to achieve long-term maintenance of organ function ex vivo. Here we outline current strategies for organ culture, or organomatics, and how these systems can be regulated by means of computational algorithms, or organometrics, to achieve the organ culture platforms anticipated in modern-day biomedicine.
High voltage, short pulsed electric fields (PEF) is a non-thermal ablation method, in which defined PEF irreversibly destabilize cell membranes, while preserving other tissue components such as the extracellular matrix (ECM). In the present report, we show that PEF ablated rat skin retains its microvascular blood supply and ECM structure. Complete regeneration of epidermis, hair follicles, sebaceous glands, and the panniculus carnosusis observed two months after the ablation. Our results clearly indicate that non-thermal PEF has the potential to be a powerful and novel tool for scarless tissue regeneration.
In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.
The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen “double gel” or “overlay”. To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.
Tissue-engineered in vitro models have the potential to be used for investigating inflammation in the complex microenvironment found in vivo. We have developed an in vitro model of hepatic tissue that facilitates real-time monitoring of endothelium activation in liver tissue. This was achieved by creating a layered coculture model in which hepatocytes were embedded in collagen gel and a reporter clone of endothelial cells, which synthesizes green fluorescent protein in response to nuclear factor-kappa B (NF-κB) activation, was overlaid on top of the gel. The efficacy of our approach was established by monitoring in real time the dynamics of NF-κB-regulated fluorescence in response to tumor necrosis factor α. Our studies revealed that endothelial cells in coculture with hepatocytes exhibited a similar NF-κB-mediated fluorescence to both pulse and step stimulation of lipopolysaccharide. By contrast, endothelial cells in monoculture displayed enhanced NF-κB-regulated fluorescence to step in comparison to pulse lipopolysaccharide stimulation. The NF-κB-mediated fluorescence correlated with endothelial cell expression of NF-κB-regulated genes such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-Selectin, as well as with leukocyte adhesion. These findings suggest that our model provides a powerful platform for investigating hepatic endothelium activation in real time.
Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our bioartificial liver (BAL) device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL-1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL device to evaluate their efficacy in treating D-galactosamine (GalN)-induced FHF in a rat model. After extracorporeal perfusion with the BAL device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) and cytokines (IL-1 and IL-6), indicating a beneficial effect. Animal survival was significantly improved in the treated group compared to the control group. These experiments demonstrate that combining inflammatory cytokine blockade with a functional BAL device may be an effective therapeutic option in the treatment of FHF.
Dendritic cells (DCs) play a pivotal role in immune modulation. Therefore, understanding and regulating the mechanism of DC activation is paramount for functional optimization of any immunotherapy strategy. In particular, the paradoxical ability of DCs to secrete the immune suppressive enzyme indoleamine 2, 3-dioxygenase (IDO) and the suppressive cytokine IL-10 during the course of, and in response to, stimulation is of great interest. 1-Methyl-Tryptophan (1 MT) is a known inhibitor of IDO and has thus been administered in numerous in vitro and in vivo systems to block IDO activity. However, the effect 1 MT has on DCs beyond inhibiting IDO, especially in therapeutic models, has rarely been analyzed. In the current study, we have administered 1 MT via a nanopolymer-based delivery system in conjunction with an antigen (ovalbumin, OVA) and an adjuvant (CpG motif DNA) to determine both the effects of 1 MT on DCs and the resulting efficacy of the polymer-based treatments. 1 MT delivery alone, either via the polymer-based delivery vehicle or dissolved in solution, induced no significant change in DC activation as measured by surface expression of CD80, CD86, and MHCII and several secreted products such as IL-12. These same factors were upregulated however, when 1 MT was delivered in conjunction with OVA and CpG. Although soluble delivery of these components increased the levels of expression and secretion of key proteins, a differential effect of DC stimulation was seen as a result of the polymer delivery system. The T cell suppressive IL-10 secretion was lower with the polymer-based treatments and IL-12 immune-enhancing secretion was increased when 1 MT was supplemented into the polymer system. As a result, including 1 MT in the polymers along with OVA and CpG was seen to have additional effects on DC stimulation and was able to shift DCs to a state more indicative of inducing a Th1-type response.
Dendritic cells; DC; PLGA; 1-methyl-tryptophan; 1MT
Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.
The current state of the art for linear optimization in Flux Balance Analysis has been limited to single objective functions. Since mammalian systems perform various functions, a multiobjective approach is needed when seeking optimal flux distributions in these systems. In most of the available multiobjective optimization methods, there is a lack of understanding of when to use a particular objective, and how to combine and/or prioritize mutually competing objectives to achieve a truly optimal solution. To address these limitations we developed a soft constraints based linear physical programming-based flux balance analysis (LPPFBA) framework to obtain a multiobjective optimal solutions. The developed framework was first applied to compute a set of multiobjective optimal solutions for various pairs of objectives relevant to hepatocyte function (urea secretion, albumin, NADPH, and glutathione syntheses) in bioartificial liver systems. Next, simultaneous analysis of the optimal solutions for three objectives was carried out. Further, this framework was utilized to obtain true optimal conditions to improve the hepatic functions in a simulated bioartificial liver system. The combined quantitative and visualization framework of LPPFBA is applicable to any large-scale metabolic network system, including those derived by genomic analyses.
Bioartificial Liver; Hepatocytes/Linear Physical Programming; Metabolic Networks; Multiobjective Optimization; Pareto Optimality