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1.  Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells 
Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of non-parenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for ∼80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell–cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10–15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.
PMCID: PMC4382924  PMID: 25233394
2.  Surgical Models of Roux-en-Y Gastric Bypass Surgery and Sleeve Gastrectomy in Rats and Mice 
Nature protocols  2015;10(3):495-507.
Bariatric surgery is the only definitive solution currently available for the present obesity pandemic. These operations typically involve reconfiguration of gastrointestinal tract anatomy and impose profound metabolic and physiological benefits, such as substantially reducing body weight and ameliorating type II diabetes. Therefore, animal models of these surgeries offer unique and exciting opportunities to delineate the underlying mechanisms that contribute to the resolution of obesity and diabetes. Here we describe a standardized procedure for mouse and rat models of Roux-en-Y gastric bypass (80–90 minutes operative time) and sleeve gastrectomy (30–45 minutes operative time), which, to a high degree resemble operations in human. We also provide detailed protocols for both pre- and post-operative techniques that ensure a high success rate in the operations. These protocols provide the opportunity to mechanistically investigate the systemic effects of the surgical interventions, such as regulation of body weight, glucose homeostasis, and gut microbiome.
PMCID: PMC4476550  PMID: 25719268
3.  Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells 
Metabolites  2016;6(1):1.
Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.
PMCID: PMC4812330  PMID: 26742084
fatty liver; steatosis; defatting; beta-oxidation; mass balances; liver transplantation; hepatocytes
4.  A Novel Resolvin-Based Strategy for Limiting Acetaminophen Hepatotoxicity 
Acetaminophen (APAP)-induced hepatotoxicity is a major cause of morbidity and mortality. The current pharmacologic treatment for APAP hepatotoxicity, N-acetyl cysteine (NAC), targets the initial metabolite-driven injury but does not directly affect the host inflammatory response. Because of this, NAC is less effective if given at later stages in the disease course. Resolvins, a novel group of lipid mediators shown to attenuate host inflammation, may be a therapeutic intervention for APAP hepatotoxicity.
The temporal patterns of liver injury and neutrophil activation were investigated in a murine model of APAP hepatotoxicity. In addition, the effect of neutrophil depletion and resolvin administration on the severity of liver injury induced by APAP was studied. In vitro studies to investigate the mechanism of resolvin effect on hepatocyte injury and neutrophil adhesion were performed.
We demonstrate that hepatic neutrophil activation occurs secondary to the initial liver injury induced directly by APAP. We also show that neutrophil depletion attenuates APAP-induced liver injury, and administration of resolvins hours after APAP challenge not only attenuates liver injury, but also extends the therapeutic window eightfold compared to NAC. Mechanistic in vitro analysis highlights resolvins' ability to inhibit neutrophil attachment to endothelial cells in the presence of the reactive metabolite of APAP.
This study highlights the ability of resolvins to protect against APAP-induced liver injury and extend the therapeutic window compared to NAC. Although the mechanism for resolvin-mediated hepatoprotection is likely multifactorial, inhibition of neutrophil infiltration and activation appears to play an important role.
PMCID: PMC4822092  PMID: 26986653
5.  Development of a low-volume, highly sensitive microimmunoassay using computational fluid dynamics-driven multiobjective optimization 
Microfluidics and nanofluidics  2014;18(2):199-214.
Immunoassays are one of the most versatile and widely performed biochemical assays and, given their selectivity and specificity, are used in both clinical and research settings. However, the high cost of reagents and relatively large sample volumes constrain the integration of immunoassays into many applications. Scaling the assay down within microfluidic devices can alleviate issues associated with reagent and sample consumption. However, in many cases a new device is designed and empirically optimized for each specific analyte, a costly and time consuming approach. In this paper, we report the development of a microfluidic bead-based immunoassay which, using antibody coated microbeads, can potentially detect any analyte or combination of analytes for which antibody coated microbeads can be generated. We also developed a computational reaction model and optimization algorithm that can be used to optimize the device for any analyte. We applied this technique to develop a low volume IL-6 immunoassay with high sensitivity (358 fM, 10 pg/mL) and a large dynamic range (4 orders of magnitude). This device design and optimization technique can be used to design assays for any protein with an available antibody and can be used with a large number of applications including biomarker discovery, temporal in vitro studies using a reduced number of cells and reagents, and analysis of scarce biological samples in animal studies and clinical research settings.
PMCID: PMC4327895  PMID: 25691853
Immunoassay; Microfluidic; Computational Fluid Dynamics; Multi-Objective Optimization
6.  Rat liver regeneration following ablation with irreversible electroporation 
PeerJ  2016;4:e1571.
During the past decade, irreversible electroporation (IRE) ablation has emerged as a promising tool for the treatment of multiple diseases including hepatic cancer. However, the mechanisms behind the tissue regeneration following IRE ablation have not been investigated. Our results indicate that IRE treatment immediately kills the cells at the treatment site preserving the extracellular architecture, in effect causing in vivo decellularization. Over the course of 4 weeks, progenitor cell differentiation, through YAP and notch pathways, together with hepatocyte expansion led to almost complete regeneration of the ablated liver leading to the formation of hepatocyte like cells at the ablated zone. We did not observe significant scarring or tumor formation at the regenerated areas 6 months post IRE. Our study suggests a new model to study the regeneration of liver when the naïve extracellular matrix is decellularized in vivo with completely preserved extracellular architecture.
PMCID: PMC4727979  PMID: 26819842
Liver regeneration; Irreversible electroporation; Liver ablation; Pulsed electric fields; Scarless regeneration; Progenitor cells; Hepatocytes
7.  Real-time Needle Steering in Response to Rolling Vein Deformation by a 9-DOF Image-Guided Autonomous Venipuncture Robot 
Venipuncture is the most common invasive medical procedure performed in the United States and the number one cause of hospital injury. Failure rates are particularly high in pediatric and elderly patients, whose veins tend to deform, move, or roll as the needle is introduced. To improve venipuncture accuracy in challenging patient populations, we have developed a portable device that autonomously servos a needle into a suitable vein under image guidance. The device operates in real time, combining near-infrared and ultrasound imaging, computer vision software, and a 9 degrees-of-freedom robot that servos the needle. In this paper, we present the kinematic and mechanical design of the latest generation robot. We then investigate in silico and in vitro the mechanics of vessel rolling and deformation in response to needle insertions performed by the robot. Finally, we demonstrate how the robot can make real-time adjustments under ultrasound image guidance to compensate for subtle vessel motions during venipuncture.
PMCID: PMC4714798  PMID: 26779381
8.  Low Power Laser Irradiation Stimulates the Proliferation of Adult Human Retinal Pigment Epithelial Cells in Culture 
We investigated the effects of low power laser irradiation on the proliferation of retinal pigment epithelial (RPE) cells. Adult human RPE cells were artificially pigmented by preincubation with sepia melanin, and exposed to a single sublethal laser pulse (590 nm, 1 µs, <200 mJ/cm2). DNA synthesis, cell number, and growth factor activity in irradiated RPE cells were subsequently monitored. The effect of sublethal laser irradiation on the “wound” healing response of an RPE monolayer in an in vitro scratch assay was also investigated. Single pulsed laser irradiation increased DNA synthesis in pigmented RPE cells measured 6 h post-treatment. In the scratch assay, laser irradiation increased the rates of cell proliferation and wound closure. Conditioned medium, collected 48 h following laser treatment, increased cell proliferation of unirradiated cells. Irradiation increased RPE cell secretion of platelet-derived growth factor (PDGF)-B chain, and increased mRNA levels of several growth factors and their receptors, including PDGF, transforming growth factor-β1, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, as well as heat shock proteins. This demonstrates, for the first time, that low power single pulsed laser irradiation stimulates the proliferation of RPE cells, and upregulates growth factors that are mitogenic for RPE cells.
PMCID: PMC4698896  PMID: 26740823
Photothermolysis; Wound healing; Heat shock; Macular degeneration
9.  Pulsed Electric Fields for Burn Wound Disinfection in a Murine Model 
Emerging bacterial resistance renders many antibiotics ineffective, making alternative strategies of wound disinfection important. Here the authors report on a new, physical burn wound disinfection method: pulsed electric fields (PEFs). High voltage, short PEFs create nonthermal, permanent damage to cell membranes, possibly by irreversible electroporation. In medicine, PEF technology has recently been used for nonthermal ablation of solid tumors. The authors have expanded the spectrum of PEF applications in medicine to burn wound disinfection. A third-degree burn was induced on the dorsal skin of C57BL/6 mice. Immediately after the injury, the burn wound was infected with Acinetobacter baumannii expressing the luxCDABE operon. Thirty minutes after infection, the infected areas were treated with 80 pulses delivered at 500 V/mm, 70 μs, 1 Hz. The authors used bioluminescence to quantify bacteria on skin. Three animals were used for each experimental condition. PEFs were effective in the disinfection of infected burned murine skin. The bacterial load reduction correlated with the number of delivered pulses. Forty pulses of 500 V/mm led to a 2.04 ± 0.29 Log10 reduction in bacterial load; 80 pulses led to the immediate 5.53 ± 0.30 Log10 reduction. Three hours after PEF, the bacterial reduction of the skin treated with 500 V/mm, 80 pulses was 4.91 ± 0.71 Log10. The authors introduce a new method of wound disinfection using high voltage, short PEFs. They believe that PEF technology may represent an important alternative to antibiotics in addressing bacterial contamination of wounds, particularly those contaminated with multidrug-resistant bacteria.
PMCID: PMC4286470  PMID: 25167374
10.  An automated image processing method to quantify collagen fiber organization within cutaneous scar tissue 
Experimental dermatology  2014;24(1):78-80.
Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodeling process. We have recently developed an image analysis technique for the rapid quantification of fiber alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fiber density and directional variance from Masson’s Trichrome stained sections for quantifying changes in collagen organization during scar remodeling. Significant increases in collagen fiber density are detectable soon after burn injury in a rat model. Decreased fiber directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fiber alignment. This automated analysis of fiber organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration.
PMCID: PMC4289465  PMID: 25256009
Collagen; fiber alignment; burns; scar remodeling; image analysis
11.  Retinal Pigment Epithelium Differentiation of Stem Cells: Current Status and Challenges 
Degeneration and loss of retinal pigment epithelium (RPE) is the cause of a number of degenerative retinal diseases, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, leading to blindness that affects three million Americans as of now. Transplantation of RPE aims to restore retinal structure and the interaction between the RPE and photoreceptors, which is fundamental to sight. Although a significant amount of progress has been made in the past 20 years in autologous RPE transplantation, sources for RPE cells are limited. Recent advances in stem cell culture and differentiation techniques have allowed the generation of RPE cells from pluripotent stem cells. In this review, we discuss strategies for generating functional RPE cells from human embryonic stem cells and induced pluripotent stem cells, and summarize transplantation studies of these derived RPEs. We conclude with challenges in cell-replacement therapies using human embryonic and induced pluripotent stem cell-derived RPEs.
PMCID: PMC4635560  PMID: 20528731
embryonic stem cells; adult retinal stem cells; retinal progenitor cells; retinal degeneration
12.  The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model 
PLoS ONE  2015;10(11):e0140440.
In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.
PMCID: PMC4631332  PMID: 26528713
13.  Layer-by-layer heparinization of decellularized liver matrices to reduce thrombogenicity of tissue engineered grafts 
Tissue-engineered liver grafts may offer a viable alternative to orthotopic liver transplantation and help overcome the donor organ shortage. Decellularized liver matrices (DLM) have a preserved vasculature and sustain hepatocellular function in culture, but graft survival after transplantation remains limited due to thrombogenicity of the matrix.
To evaluate the effect of heparin immobilization on DLM thrombogenicity.
Heparin was immobilized on DLMs by means of layer-by-layer deposition. Grafts with 4 or 8 bilayers and 2 or 4 g/L of heparin were recellularized with primary rat hepatocytes and maintained in culture for 5 days. Hemocompatibility of the graft was assessed by ex vivo diluted whole-blood perfusion and heterotopic transplantation.
Heparin was deposited throughout the matrix and the heparin content in the graft was higher with increasing number of bilayers and concentration of heparin. Recellularization and in vitro albumin and urea production were unaffected by heparinization. Resistance to blood flow during ex vivo perfusion was lower with increased heparinization and, macroscopically, no clots were visible in grafts with 8 bilayers. Following transplantation, flow through the graft was limited in all groups. Histological evidence of thrombosis was lower in heparinized DLMs, but transplantation of DLM grafts was not improved.
Layer-by-layer deposition of heparin on a DLM is an effective method of immobilizing heparin throughout the graft and does not impede recellularization or hepatocellular function in vitro. Thrombogenicity during ex vivo blood perfusion was reduced in heparinized grafts and optimal with 8 bilayers, but transplantation remained unsuccessful with this method.
Relevance for patients
Tissue engineered liver grafts may offer a viable solution to dramatic shortages in donor organs
PMCID: PMC4607069  PMID: 26478914
Tissue engineering; decellularization; recellularization; heparinization; thrombogenicity; hemocompatibility; transplantation
14.  Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability 
Emerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism.
A meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells.
Nonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-α (TNFα) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNFα production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNFα in altering intestinal permeability, we found that mice genetically resistant to TNFα-myosin light chain kinase (MLCK)–induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury.
Our clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNFα and MLCK.
PMCID: PMC4578658  PMID: 26405687
Meta-Analysis; Myosin Light Chain Kinase; Steatosis; Tight Junctions
15.  Elevated Sensitivity of Macrosteatotic Hepatocytes to Hypoxia-Reoxygenation Stress Is Reversed by a Novel Defatting Protocol 
Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) content in the form of large lipid droplets (LDs), reduced ATP, and elevated reactive oxygen species (ROS), contributing to their elevated sensitivity to ischemia-reperfusion injury during transplantation. Decreasing macrosteatosis in living donors through dieting has been shown to improve transplantation outcome. Accomplishing the same feat in deceased donor grafts would require ex-vivo exposure to potent defatting agents. Herein, we used a rat hepatocyte culture system exhibiting macrosteatotic LD morphology, elevated TG levels, and elevated sensitivity to hypoxia and reoxygenation (H/R), to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48h with a defatting cocktail, previously developed to promote TG catabolism, reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but did not ameliorate sensitivity to H/R. L-carnitine supplementation to this cocktail, together with hyperoxic exposure, yielded a similar reduction in macrosteatotic LD numbers, and to a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl-CoA to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27%, nearing ATP levels of lean controls, and completely abolished H/R sensitivity as indicated by ~85% viability post H/R and return of cytosolic lactate dehydrogenase release down to levels seen in lean controls. Cultures maintained for 48h post H/R were ~83% viable and exhibited superior urea secretion and bile canalicular transport compared to untreated macrosteatotic cultures. These findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, suggesting a possible route for the recovery of discarded macrosteatotic grafts.
PMCID: PMC4117728  PMID: 24802973
Hypoxia; macrosteatosis; hyperoxia; carnitine; ATP; ROS
16.  Effects of Burn Injury on Markers of Hypermetabolism in Rats 
The basic metrics of hypermetabolism have not been thoroughly characterized in rat burn injury models. We examined three models expected to differ in sensitivity to burn injury to identify that which group(s) exhibited the most clinically relevant metabolic response. Six and 12 weeks old male CD (6 week mCD and 12 week mCD) rats, and 12 weeks old female Fischer (12 week fFI) rats received a 20% total body surface area burn, followed by saline resuscitation. Activity, core body temperature, heart rate (via implanted telemetry devices), body weight, food and water intake, and fecal output were measured daily for 1 week before and after burn. Rats lost weight initially postburn but resumed weight gain by 1 week, except for 12 week mCD rats. Core body temperature increased above normal 2 days postburn and returned to baseline by 1 week. Food intake, normalized to body weight, remained unchanged postburn for 12 week mCD rats, but decreased in 6 week mCD rats and increased in 12 week fFI rats. Heart rate in the 12 week mCD and 12 week fFI rats remained at 10 to 15% above baseline, whereas, in 6 week mCD, heart rates returned to baseline after 4 days. Activity levels were unchanged for 12 week fFI and 6 week mCD rats postburn, but decreased for 12 week mCD rats. Postburn hypermetabolism was most significant and sustained in 12 week mCD rats, of least consequence and brief in 6 week mCD rats, and intermediate in 12 week fFI rats. The disparate responses indicate that the choice of animal model should be carefully considered in hypermetabolism studies.
PMCID: PMC4500046  PMID: 19898103
17.  Apolipoprotein B–Dependent Hepatitis C Virus Secretion Is Inhibited by the Grapefruit Flavonoid Naringenin 
Hepatology (Baltimore, Md.)  2008;47(5):1437-1445.
Hepatitis C virus (HCV) infects over 3% of the world population and is the leading cause of chronic liver disease worldwide. HCV has long been known to associate with circulating lipoproteins, and its interactions with the cholesterol and lipid pathways have been recently described. In this work, we demonstrate that HCV is actively secreted by infected cells through a Golgi-dependent mechanism while bound to very low density lipoprotein (vLDL). Silencing apolipoprotein B (ApoB) messenger RNA in infected cells causes a 70% reduction in the secretion of both ApoB-100 and HCV. More importantly, we demonstrate that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, inhibits the microsomal triglyceride transfer protein activity as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and acyl-coenzyme A:cholesterol acyltransferase 2 in infected cells. Stimulation with naringenin reduces HCV secretion in infected cells by 80%. Moreover, we find that naringenin is effective at concentrations that are an order of magnitude below the toxic threshold in primary human hepatocytes and in mice.
These results suggest a novel therapeutic approach for the treatment of HCV infection.
PMCID: PMC4500072  PMID: 18393287
18.  Supercooling Preservation Of The Rat Liver For Transplantation 
Nature protocols  2015;10(3):484-494.
The current standard for liver preservation is limited in duration. Employing a novel subzero preservation technique that includes supercooling and machine perfusion can significantly improve preservation and prolong storage times. By loading rat livers with cryoprotectants to prevent both intra- and extracellular ice formation and protect against hypothermic injury, livers can be cooled to −6 °C without freezing and kept viable for up to 96 hours. Here, we describe the procedures of loading cryoprotectants by means of subnormothermic machine perfusion (SNMP), controlled cooling to a supercooled state, followed by SNMP recovery and orthotopic liver transplantation.
PMCID: PMC4494653  PMID: 25692985
19.  Enhanced Differentiation of Embryonic Stem Cells Using Co-Cultivation With Hepatocytes 
Biotechnology and bioengineering  2008;101(6):1332-1343.
We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257–266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced “globally” within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.
PMCID: PMC4492440  PMID: 18571804
embryonic stem cell; liver; E-cadherin; co-culture; hepatocyte; differentiation
20.  Solving Medical Problems with BioMEMS 
IEEE pulse  2011;2(6):51-59.
PMCID: PMC4490689  PMID: 22147069
Biotechnology and bioengineering  2009;102(5):1466-1474.
Fatty liver disease is a problem of growing clinical importance due to its association with the increasingly prevalent conditions of obesity and diabetes. While steatosis represents a reversible state of excess intrahepatic lipid, it is also associated with increased susceptibility to oxidative and cytokine stresses and progression to irreversible hepatic injury characterized by steatohepatitis, cirrhosis, and malignancy. Currently, the molecular mechanisms underlying progression of this dynamic disease remain poorly understood, particularly at the level of transcriptional regulation. We recently constructed a library of stable monoclonal green fluorescent protein (GFP) reporter cells that enable transcriptional regulation to be studied dynamically in living cells. Here, we adapt the reporter cells to create a model of steatosis that will allow investigation of transcriptional dynamics associated with the development of steatosis and the response to subsequent “second hit” stresses. The reporter model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, increased reactive oxygen species accumulation, decreased mitochondrial membrane potential, increased susceptibility to apoptotic cytokine stresses, and decreased proliferation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor κB (NFκB), heat shock response element (HSE), and glucocorticoid response element (GRE) in response to their classical inducers under lean and fatty conditions and found that intracellular lipid accumulation was associated with dose-dependent impairment of NFκB and HSE but not GRE activation. Thus, steatotic reporter cells represent an efficient model for studying transcriptional responses and have the potential to provide important insights into the progression of fatty liver disease.
PMCID: PMC4490792  PMID: 19061238
steatosis; reporter cells; in vitro disease model
22.  A New Technique for Primary Hepatocyte Expansion In Vitro 
Biotechnology and bioengineering  2008;101(2):345-356.
The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell–cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell–cell interactions in the modulation of hepatic proliferation and function.
PMCID: PMC4487520  PMID: 18465801
hepatocyte proliferation; coculture; DNA synthesis; cell patterning; subculture
23.  Augmentation of EB-Directed Hepatocyte-Specific Function via Collagen Sandwich and SNAP 
Biotechnology progress  2008;24(5):1132-1141.
The development of implantable engineered liver tissue constructs and ex vivo hepatocyte-based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)-mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin-18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin-M (OSM) or S-nitroso-N-acetylpenicillamine (SNAP) supplementation of EB-derived hepatocyte-lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long-term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver-specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte-like function. Maintenance and augmentation of hepatocyte-like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.
PMCID: PMC4487523  PMID: 19194923
ES cells; hepatocytes; collagen sandwich; SNAP; cytochrome P450
24.  Transient Gene Delivery for Functional Enrichment of Differentiating Embryonic Stem Cells 
Biotechnology and bioengineering  2008;101(5):859-872.
There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. We have reported previously a variety of methods for differentiating murine embryonic stem (ES) cells into hepatocyte-like cells. One major challenge of our work and others in the field has been the ability to selectively purify and enrich these cells from a heterogeneous population. Traditional approaches for inserting new genes (e.g., stable transfection, knock-in, retroviral transduction) involve permanent alterations in the genome. These approaches can lead to mutations and involve the extra costs and time of developing, validating and maintaining new cell lines. We have developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells are purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis is performed. In an effort to test the ability of the reporters to work in a transient environment for our differentiation system, we engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection efficiency of delivering these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. Development of gene reporter systems that allow us to identify, purify and assess homogeneous populations of cells is important in better understanding stem cell differentiation pathways. And engineering cellular systems without making permanent gene changes will be critical for the generation of clinically acceptable cellular material in the future.
PMCID: PMC4487524  PMID: 18942772
stem cell differentiation; gene delivery; transient transfection; cell sorting; Cyp450 detoxification activity
25.  Portable robot for autonomous venipuncture using 3D near infrared image guidance 
Technology  2013;1(1):72-87.
Venipuncture is pivotal to a wide range of clinical interventions and is consequently the leading cause of medical injury in the U.S. Complications associated with venipuncture are exacerbated in difficult settings, where the rate of success depends heavily on the patient's physiology and the practitioner's experience. In this paper, we describe a device that improves the accuracy and safety of the procedure by autonomously establishing a peripheral line for blood draws and IV's. The device combines a near-infrared imaging system, computer vision software, and a robotically driven needle within a portable shell. The device operates by imaging and mapping in real-time the 3D spatial coordinates of subcutaneous veins in order to direct the needle into a designated vein. We demonstrate proof of concept by assessing imaging performance in humans and cannulation accuracy on an advanced phlebotomy training model.
PMCID: PMC4482475  PMID: 26120592

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