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1.  Perspectives on Non-Animal Alternatives for Assessing Sensitization Potential in Allergic Contact Dermatitis 
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.
doi:10.1007/s12195-011-0189-4
PMCID: PMC3985137  PMID: 24741377
Immunobiology; Skin sensitization; In silico approaches; QSAR; 2D cell based models; 3D skin tissue models; Integrated Testing Strategies; Microfabrication; Microfluidics
2.  Non-thermal, pulsed electric field cell ablation: A novel tool for regenerative medicine and scarless skin regeneration 
Technology  2013;1(1):1-8.
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.
doi:10.1142/S233954781320001X
PMCID: PMC4078877
3.  Target DNA detection and quantitation on a single cell with single base resolution 
Technology  2013;1(1):88-.
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.
doi:10.1142/S2339547813500088
PMCID: PMC4073798  PMID: 24977169
4.  A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture 
Technology  2014;2(1):67-74.
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.
doi:10.1142/S2339547814500083
PMCID: PMC4054686  PMID: 24932459
5.  ANALYSIS OF DENDRITIC CELL STIMULATION UTILIZING A MULTI-FACETED NANOPOLYMER DELIVERY SYSTEM AND THE IMMUNE MODULATOR 1-METHYL TRYPTOPHAN 
Nano LIFE  2010;1(3-4):239-250.
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.
doi:10.1142/S1793984410000171
PMCID: PMC3998209  PMID: 24772192
Dendritic cells; DC; PLGA; 1-methyl-tryptophan; 1MT
6.  Stem Cells for Liver Repopulation 
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.
Recent findings
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.
Summary
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.
doi:10.1097/MOT.0b013e3283328070
PMCID: PMC3725723  PMID: 19779345
Hepatocyte transplantation; xenogenic hepatocytes; stem cell-derived hepatocytes; liver tissue engineering
7.  Optimality and thermodynamics determine the evolution of transcriptional regulatory networks† 
Molecular bioSystems  2011;8(2):511-530.
Transcriptional motifs are small regulatory interaction patterns that regulate biological functions in highly-interacting cellular networks. Recently, attempts have been made to explain the significance of transcriptional motifs through dynamic function. However, fundamental questions remain unanswered. Why are certain transcriptional motifs with similar dynamic function abundant while others occur rarely? What are the criteria for topological generalization of these motifs into complex networks? Here, we present a novel paradigm that combines non-equilibrium thermodynamics with multiobjective-optimality for network analysis. We found that energetic cost, defined herein as specific dissipation energy, is minimal at the optimal environmental conditions and it correlates inversely with the abundance of the network motifs obtained experimentally for E. coli and S. cerevisiae. This yields evidence that dissipative energetics is the underlying criteria used during evolution for motif selection and that biological systems during transcription tend towards evolutionary selection of subgraphs which produces minimum specific heat dissipation under optimal conditions, thereby explaining the abundance/rare occurrence of some motifs. We show that although certain motifs had similar dynamical functionality, they had significantly different energetic cost, thus explaining the abundance/rare occurrence of these motifs. The presented insights may establish global thermodynamic analysis as a backbone in designing and understanding complex networks systems, such as metabolic and protein interaction networks.
doi:10.1039/c1mb05177f
PMCID: PMC3964374  PMID: 22076617
8.  Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells 
PLoS ONE  2014;9(2):e89882.
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.
doi:10.1371/journal.pone.0089882
PMCID: PMC3931832  PMID: 24587097
9.  AN ORGANOTYPIC UNIAXIAL STRAIN MODEL USING MICROFLUIDICS 
Lab on a chip  2013;13(3):10.1039/c2lc41063j.
Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections.
doi:10.1039/c2lc41063j
PMCID: PMC3546521  PMID: 23233120
10.  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
11.  Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms 
Annals of biomedical engineering  2012;40(9):1851-1861.
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.
doi:10.1007/s10439-012-0555-y
PMCID: PMC3901163  PMID: 22484829
Non-viral transfection; Lentiviruses; Primary rat hepatocytes; Huh7.5.1; Transfection efficiency; Hepatic function
12.  Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation 
The Journal of surgical research  2011;175(1):10.1016/j.jss.2011.03.003.
Background
Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C.
Methods
Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls.
Results
Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values.
Conclusions
MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of subnormothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.
doi:10.1016/j.jss.2011.03.003
PMCID: PMC3863393  PMID: 21550058
liver transplantation; reperfusion injury; sub-normothermic machine perfusion
13.  MICROFLUIDIC DEVICE FOR EXAMINING DIRECTIONAL SENSING IN DENDRITIC CELL CHEMOTAXIS 
Nano LIFE  2012;2(2):1250011.
Dendritic cell chemotaxis is an important process involved in the acquisition of adaptive immunity. Despite several studies, our understanding of this process remains limited. One of the reasons for this is the lack of experimental models that give us real-time information on dendritic cell locomotion. Here, using tools in microfluidics, we have fabricated a microdevice that allows us to monitor dendritic cell migration in a chemokine gradient in real time. We successfully observed the migration of dendritic cells derived from a myeloid leukemia cell line (MUTZ-3) in a soluble chemokine (CCL-19) gradient. Our experiments suggest the utility of microdevices in monitoring dendritic cell chemotaxis in real time and getting important information regarding migration speeds and distances previously not available from conventional chemotaxis assays. This kind of data is useful for building mechanistic mathematical models of dendritic cell chemotaxis that may give us novel insights to the process of dendritic cell chemotaxis.
doi:10.1142/S1793984411000475
PMCID: PMC3782312  PMID: 24073020
Chemotaxis; dendritic cells; BioMEMs; migration; CCL-19
14.  A Mesenchymal Stem Cell Potency Assay 
Mesenchymal stem cells (MSCs) are capable of modulating the immune system and have been used to successfully treat a variety of inflammatory diseases in preclinical studies. Recent evidence has implicated paracrine signaling as the predominant mechanism of MSC therapeutic activity. We have shown in models of inflammatory organ failure that the factors secreted by MSCs are capable of enhancing survival, downregulating inflammation, and promoting endogenous repair programs that lead to the reversal of these diseases. As a marker of disease resolution, we have observed an increase in serum IL-10 when MSC-conditioned medium (MSC-CM) or lysate (MSC-Ly) is administered in vivo. Here we present an in vitro model of IL-10 release from blood cells that recapitulates this in vivo phenomenon. This assay provides a powerful tool in analyzing the potency of MSC-CM and MSC-Ly, as well as characterizing the interaction between MSC-CM and target cells in the blood.
doi:10.1007/978-1-60761-869-0_16
PMCID: PMC3759509  PMID: 20941614
Mesenchymal stem cell; IL-10; Potency assay; Organ injury; Inflammation; Autoimmunity; Transplantation
15.  Embryoid Body–Mediated Differentiation of Mouse Embryonic Stem Cells Along a Hepatocyte Lineage: Insights from Gene Expression Profiles 
Tissue engineering  2006;12(6):1515-1525.
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.
doi:10.1089/ten.2006.12.1515
PMCID: PMC3199957  PMID: 16846348
16.  Resolvin D2 prevents secondary thrombosis and necrosis in a mouse burn wound model 
Deep partial thickness burns are subject to delayed necrosis of initially viable tissues surrounding the primary zone of thermally induced coagulation, which results in an expansion of the burn wound, both in area and depth, within 48 hours postburn. Neutrophil sequestration and activation leading to microvascular damage is thought to mediate this secondary tissue damage. Resolvins, a class of endogenous mediators derived from omega-3 polyunsaturated fatty acids, have been shown to regulate the resolution of inflammation. We hypothesized that exogenous resolvins could mitigate the deleterious impact of the inflammatory response in burn wounds. Using two different mouse burn injury models involving significant partial thickness injuries, we found that a systemically administered single dose of resolvin D2 (RvD2) as low as 25 pg/g bw given within an interval of up to 4 hours postburn effectively prevented thrombosis of the deep dermal vascular network and subsequent dermal necrosis. By preserving the microvascular network, RvD2 enhanced neutrophil access to the dermis, but prevented neutrophil-mediated damage through other anti-inflammatory actions, including inhibition of tumor necrosis factor-α, interleukin-1β, and neutrophil platelet–endothelial cell adhesion molecule-1. In a clinical context, RvD2 may be therapeutically useful by reducing the need for surgical debridement and the area requiring skin grafting.
doi:10.1111/j.1524-475X.2012.00853.x
PMCID: PMC3746505  PMID: 23110665
17.  Soft Constraints-Based Multiobjective Framework forFlux Balance Analysis 
Metabolic engineering  2010;12(5):429-445.
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.
doi:10.1016/j.ymben.2010.05.003
PMCID: PMC2922426  PMID: 20553945
Bioartificial Liver; Hepatocytes/Linear Physical Programming; Metabolic Networks; Multiobjective Optimization; Pareto Optimality
18.  Layered patterning of hepatocytes in co-culture systems using microfabricated stencils 
BioTechniques  2010;48(1):47-52.
Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 µm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.
doi:10.2144/000113317
PMCID: PMC3147300  PMID: 20078427
hepatocytes; co-culture; layered cell patterning; cellular interactions; fibroblasts
19.  Application of whole-organ tissue engineering in hepatology 
Initially hailed as the ultimate solution to organ failure, engineering of vascularized tissues such as the liver has stalled because of the need for a well-structured circulatory system that can maintain the cells seeded inside the construct. A new approach has evolved to overcome this obstacle. Whole-organ decellularization is a method that retains most of the native vascular structures of the organ, providing microcirculatory support and structure, which can be anastomosed with the recipient circulation. The technique was first applied to the heart and then adapted for the liver. Several studies have shown that cells can be eliminated, the extracellular matrix and vasculature are reasonably preserved and, after repopulation with hepatocytes, these grafts can perform hepatic functions in vitro and in vivo. Progress is rapidly being made as researchers are addressing several key challenges to whole-organ tissue engineering, such as ensuring correct cell distribution, nonparenchymal cell seeding, blood compatibility, immunological concerns, and the source of cells and matrices.
doi:10.1038/nrgastro.2012.140
PMCID: PMC3732057  PMID: 22890112
20.  Resuscitation of Ischemic Donor Livers with Normothermic Machine Perfusion: A Metabolic Flux Analysis of Treatment in Rats 
PLoS ONE  2013;8(7):e69758.
Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.
doi:10.1371/journal.pone.0069758
PMCID: PMC3724866  PMID: 23922793
21.  Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes 
PLoS ONE  2013;8(7):e69334.
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.
doi:10.1371/journal.pone.0069334
PMCID: PMC3713052  PMID: 23874947
22.  Excorporeal Normothermic Machine Perfusion Resuscitates Pig DCD Livers with Extended Warm Ischemia 
The Journal of surgical research  2011;173(2):e83-e88.
Background
The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system.
Methods
Livers from 12 Yorkshire pigs (20–30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly.
Results
Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts.
Conclusion
Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.
doi:10.1016/j.jss.2011.09.057
PMCID: PMC3682784  PMID: 22099594
donor after cardiac death (DCD); normothermic machine perfusion (NMP); warm ischemia; transplantation; ATP
23.  Bone Marrow Mesenchymal Stromal Cells Attenuate Organ Injury Induced by LPS and Burn 
Cell transplantation  2010;19(6):823-830.
Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury-models induced by Lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.
doi:10.3727/096368910X508942
PMCID: PMC2957544  PMID: 20573305
anti-inflammation; anti-apoptosis; sepsis; burn; lipopolysaccharide (LPS); systemic inflammatory response syndrome (SIRS); and multiple organ injury
24.  PLGA-polymer encapsulating tumor antigen and CpG DNA administered into the tumor microenvironment elicits a systemic antigen-specific IFN-γ response and enhances survival 
Journal of cancer therapy  2013;4(1):280-290.
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.
doi:10.4236/jct.2013.41035
PMCID: PMC3670804  PMID: 23741626
Cytokine; Immune response; Immunomodulation; Macrophage; Microencapsulation; Vaccine
25.  Metabolic Preconditioning of Donor Organs 
Metabolic engineering  2009;11(4-5):274.
Fatty liver is a significant risk factor for liver transplantation, and accounts for nearly half of the livers rejected from the donor pool. We hypothesized that metabolic preconditioning via ex vivo perfusion of the liver graft can reduce fat content and increase post-transplant survival to an acceptable range. We describe a perfusate medium containing agents that promote the defatting of hepatocytes and explanted livers. Defatting agents were screened on cultured hepatocytes made fatty by pre-incubation with fatty acids. The most effective agents were then used on fatty livers. Fatty livers were isolated from obese Zucker rats and normothermically perfused with medium containing a combination of defatting agents. This combination decreased the intracellular lipid content of cultured hepatocytes by 35% over 24 hours, and of perfused livers by 50% over 3 hours. Metabolite analysis suggests that the defatting cocktail upregulated both lipid oxidation and export. Furthermore, gene expression analysis for several enzymes and transcription factors involved in fatty acid oxidation and triglyceride clearance were elevated. We conclude that a cocktail of defatting agents can be used to rapidly clear excess lipid storage in fatty livers, thus providing a new means to recondition donor livers deemed unacceptable or marginally acceptable for transplantation.
doi:10.1016/j.ymben.2009.05.005
PMCID: PMC2814076  PMID: 19508897
nuclear receptors; normothermic perfusion; hepatocytes; steatosis; liver transplantation

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