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One of the fundamental challenges facing the development of new chemical entities within the pharmaceutical industry is the extrapolation of key in vivo parameters from in vitro cell culture assays and animal studies. Development of microscale devices and screening assays incorporating primary human cells can potentially provide better, faster and more efficient prediction of in vivo toxicity and clinical drug performance. With this goal in mind, large strides have been made in the area of microfluidics to provide in vitro surrogates that are designed to mimic the physiological architecture and dynamics. More recent advancements have been made in the development of in vitro analogues to physiologically-based pharmacokinetic (PBPK) models - a mathematical model that represents the body as interconnected compartments specific for a particular organ. In this review we highlight recent advancements in human hepatocyte microscale culture, and describe the next generation of integrated devices, whose potential allows for the high throughput assessment of drug metabolism, distribution and pharmacokinetics.

The mechanical force exerted by a nanoactuator due to pH actuation is discussed from a statistical mechanics and free energy of conformational change viewpoint. We use molecular dynamics to show that the nanoactuator, based on the coiled-coil leucine zipper portion of a yeast transcriptional activator protein, can generate mechanical forces of the order of 20-40 pN upon pH modulation. The forces are generated due to the electrostatic repulsions at low pH between His-tag handles and other charged residues engineered into the protein sequence. The biological force output of the nanoactuator is comparable to that generated by adenosine triphosphate (ATP)-based molecular motors such as myosin and kinesin even though the nanoactuator is smaller in size to these molecular motors. The force calculation technique can readily be applied to other biomolecular systems and has implications in the area of bionanotechnology and in particular to study and characterize the properties of novel protein and DNA-based nanodevices.

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.

The liver is a major site for the metabolism of xenobiotic compounds due to its abundant level of phase I/II metabolic enzymes. With the cost of drug development escalating to over $400 million/drug there is an urgent need for the development of rigorous models of hepatic metabolism for preclinical screening of drug clearance and hepatotoxicity. Here, we present a microenvironment in which primary human and rat hepatocytes maintain a high level of metabolic competence without a long adaptation period. We demonstrate that co-cultures of hepatocytes and endothelial cells in serum-free media seeded under 95% oxygen maintain functional apical and basal polarity, high levels of cytochrome P450 activity, and gene expression profiles on par with freshly isolated hepatocytes. These oxygenated co-cultures demonstrate a remarkable ability to predict in vivo drug clearance rates of both rapid and slow clearing drugs with an R 2 of 0.92. Moreover, as the metabolic function of oxygenated co-cultures stabilizes overnight, preclinical testing can be carried out days or even weeks before other culture methods, significantly reducing associated labor and cost. These results are readily extendable to other culture configurations including three-dimensional culture, bioreactor studies, as well as microfabricated co-cultures.

Integral to the discovery of new pharmaceutical entities is the ability to predict in vivo pharmacokinetic parameters from early stage in vitro data generated prior to the onset of clinical testing. Within the pharmaceutical industry, a whole host of assay methods and mathematical models exist to predict the in vivo pharmacokinetic parameters of drug candidates. One of the most important pharmacokinetic properties of new drug candidates predicted from these methods and models is the hepatic clearance. Current methods, while useful, are still limited in their predictive efficacy. In order to address this issue, we have established a novel microfluidic in vitro culture system, the patented HmuREL device. The device comprises multiple compartments that are designed to be proportional to the physiological architectures and enhanced with the consideration of flow. Here we demonstrate the functionality of the liver-relevant chamber in the HmuREL device, and the feasibility of utilizing our system for predicting hepatic clearance. Cryopreserved human hepatocytes from a single donor were seeded within the HmuREL device to predict the in vivo hepatic clearance (CL(H)) of six marketed model compounds (carbamazepine, caffeine, timolol, sildenafil, imipramine, and buspirone). The intrinsic clearance rates from static culture controls, as well as clearance rates from the HmuREL device were subsequently compared to in vivo data available from the literature.

Cells respond to infection by sensing pathogens and communicating danger signals to noninfected neighbors; however, little is known about this complex spatiotemporal process. Here we show that activation of the innate immune system by double-stranded DNA (dsDNA) triggers intercellular communication through a gap junction-dependent signaling pathway, recruiting colonies of cells to collectively secrete antiviral and inflammatory cytokines for the propagation of danger signals across the tissue at large. By using live-cell imaging of a stable IRF3-sensitive GFP reporter, we demonstrate that dsDNA sensing leads to multicellular colonies of IRF3-activated cells that express the majority of secreted cytokines, including IFNβ and TNFα. Inhibiting gap junctions decreases dsDNA-induced IRF3 activation, cytokine production, and the resulting tissue-wide antiviral state, indicating that this immune response propagation pathway lies upstream of the paracrine action of secreted cytokines and may represent a host-derived mechanism for evading viral antiinterferon strategies.

The design hypothesis, architectures, and computational modeling of a novel peptide-based nanoactuator are presented in this paper. We engineered the α-helical coiled-coil portion of the yeast transcriptional activator peptide called GCN4 to obtain an environmentally responsive nanoactuator. The dimeric coiled-coil peptide consists of two identical approximately 4.5 nm long and approximately 3 nm wide polypeptide chains. The actuation mechanism depends on the modification of electrostatic charges along the peptide by varying the pH of the solution resulting in the reversible movement of helices and, therefore, creating the motion of an actuator. Using molecular dynamics simulations we showed that pH changes led to a reversible opening of up to 1.5 nm which is approximately 150% of the initial separation of the nanoactuator. We also investigated the forces generated by the nanoactuator upon pH actuation, using a new method based on a modified steered molecular dynamics technique. Owing to its open and close motion resembling that of tweezers, the new nanoactuator can potentially be used as a nanogripper in various nanomanipulation tasks such as detection and removal of heavy metal ions during nanofabrication processes or as a molecular switch.

Tissue-engineered models that mimic in vivo tissue organization offer the potential of capturing complex signaling pathways in vitro. In the liver, hepatocytes and endothelial cells are closely associated but separated by the extracellular matrix of the space of Disse. This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying the matrix with endothelial cells. We demonstrate that during the first few days of culture, the secretion of albumin and fibrinogen was 2-fold higher in cocultures compared to hepatocytes alone. Hepatocyte function in both cultures stabilized to a similar level during the second week, suggesting that endothelial cells can induce the early recovery of hepatocytes after isolation and seeding. Endothelial cell-conditioned medium reproduced the effect of coculture in a dose-dependent fashion, suggesting a role for endothelial cell-derived soluble factors. Endothelial cell-conditioned medium increased mRNA levels of various acute-phase proteins such as albumin, fibrinogen, transferrin, and α-macroglobulin in hepatocytes. Surprisingly, the effect of endothelial cell-conditioned medium was not mediated by growth factors or cytokines, or by secreted extracellular matrix, but by the release of the amino acid proline, which mediates endogenous collagen synthesis by hepatocytes. These findings suggest an important role for proline secretion by endothelial cells as a paracrine factor regulating hepatocyte function.—Jindal, R., Nahmias, Y., Tilles, A. W., Berthiaume, F., Yarmush, M. L. Amino acid-mediated heterotypic interaction governs performance of a hepatic tissue model.

The liver plays a major role in the early hypometabolic and later hypermetabolic phases after severe burn injury. Proteomic analysis was used to identify altered proteins in liver during these two phases. Sprague-Dawley rats were subjected to a full-thickness dorsal burn injury covering 40% of the total body surface area. Controls consisted of sham-treated animals. Liver tissues were collected on postburn days 1 and 7. The proteomic data show greater production of positive acute phase proteins on day 1 than on day 7. Many antioxidant enzymes were coordinately downregulated on day 1, including the potent biliverdin reductase. These antioxidants were restored and in some cases upregulated on day 7. This opposite trend in the change of antioxidant proteins corroborated our finding of more pronounced oxidative stress on day 1 than on day 7 as measured via protein carbonyl content. The changes of metabolic enzymes on days 1 and 7 were consistent with hypo- and hyper-metabolic states, respectively. Furthermore, a previously unreported decrease in ornithine aminotransferase on day 7 may be a key contributor to the observed increased urinary urea excretion during the hypermetabolic phase. Overall, the many differences in protein expression observed on postburn days 1 and 7 reflect the dissimilar hepatic metabolic patterns during the acute and flow phases following burn injury.

Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.

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.

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.

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. Biotechnol. Bioeng. 2008;101: 859–872. © 2008 Wiley Periodicals, Inc.

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.

Retroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 °C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme.

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.

Ionic elastin-like polypeptide (ELP) conjugates are a new class of biocompatible, self-assembling biomaterials. ELPs composed of the repeat unit (GVGVP)(n) are derived from the primary sequence of mammalian elastin and produced in Escherichia coli. These biopolymers exhibit an inverse transition temperature that renders them extremely useful for applications in cell-sheet engineering. Cationic and anionic conjugates were synthesized by the chemical coupling of ELP to polyethyleneimine (PEI) and polyacrylic acid (PAA). The self-assembly of ELP-PEI and ELP-PAA using the layer-by-layer deposition of alternately charged polyelectrolytes is a simple, versatile technique to generate bioactive and biomimetic surfaces with the ability to modulate cell-substratum interactions. Our studies are focused on cellular response to self-assembled multilayers of ionic (GVGVP)(40) incorporated within the polymeric sequence H(2)N-MVSACRGPG-(GVGVP)(40)-WP-COOH. Angle-dependent XPS studies indicated a difference in the chemical composition at the surface ( approximately 10A below the surface) and subsurface regions. These studies provided additional insight into the growth of the nanoscale multilayer assembly as well as the chemical environment that the cells can sense. Overall, cellular response was enhanced on glass substrata coated with ELP conjugates compared with uncoated surfaces. We report significant differences in cell proliferation, focal adhesions and cytoskeletal organization as a function of the number of bilayers in each assembly. These multilayer assemblies have the potential to be successfully utilized in the rational design of coatings on biomaterials to elicit a desired cellular response.

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D-galactosamine–induced rat model of acute liver injury, we show that systemic infusion of MSC-conditioned medium (MSC-CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC-CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three-fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up-regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC-CM on hepatocytes were demonstrated using in vitro assays. Conclusion: These data provide the first clear evidence that MSC-CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF. (HEPATOLOGY 2008.)

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.

Hepatitis C virus (HCV) infection is a global public health problem, affecting over 3% of the world population. HCV infection develops into a chronic condition in over 70% of the patients, ultimately leading to cirrhosis and hepatocellular carcinoma.1 Current standards of care consist of interferon (α2A) and ribavirin, which have been found to be effective in only 50% of the cases.1 However, this treatment is poorly tolerated by patients and is associated with significant side effects. Therefore, there is a pressing need for the development of alternative strategies for the treatment of HCV infection. HCV has long been known to associate with β-lipoproteins [very low density lipoprotein (vLDL) and low-density lipoprotein (LDL)] circulating in patients’ blood.2 Its E1/E2 receptors have been found to bind to both LDL and high-density lipoprotein,3 whereas HCV core protein has been shown to associate with apolipoprotein AII (ApoAII)4 and lipid droplets in HepG2 cells.5 In addition, HCV replication has been shown to be up-regulated by fatty acids and inhibited by statins; this suggests an interaction between HCV, cholesterol, and lipid metabolism.6 The recent development of an efficient cell culture system in which the full lifecycle of HCV infection is captured has opened new opportunities for the study of the viral secretion.7,8 Using this system, Gastaminza et al.9 demonstrated that intercellular HCV particles have a higher density than their secreted counterparts, suggesting that HCV might bind low-density particles prior to viral egress. Just recently, Huang et al.10 demonstrated that HCV secretion is dependent on both apolipoprotein B (ApoB) expression and vLDL assembly in a chromosomally integrated complementary DNA (cDNA) model of HCV secretion. These results strongly suggest that HCV might be “hitching a ride” along the lipoprotein lifecycle. Therefore, compounds previously shown to influence lipoprotein assembly and secretion could possibly exert a similar effect on HCV. To test this hypothesis, we used the full-length, RNA-based HCV full lifecycle model (JFH1/Huh7.5.1) previously shown to capture important aspects of viral replication, assembly, and infection. Using this model, we demonstrate that HCV is being actively secreted by infected cells in a Golgi-dependent pathway while bound to vLDL. Silencing ApoB messenger RNA (mRNA) by transfection with short hairpin RNA (shRNA) is shown to induce a 70% reduction in the secretion of ApoB, HCV core protein, and HCV RNA. More importantly, we find that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, is able to reduce HCV secretion from infected cells by 80% ± 10%. We demonstrate that naringenin inhibits ApoB secretion by inhibiting the activity of the microsomal triglyceride transfer protein (MTP) as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme reductase (HMGR) and acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2). Moreover, we find that naringenin is effective at a concentration of 200 µM, which is well below its toxic concentration for primary human hepatocytes and severe combined immunodeficient (SCID) mice.

A central problem in bioinformatics and systems biology is the selection of appropriate models in a rational and systematic way. This fundamentally combinatorial problem can be readily formulated and addressed within an integer optimization framework. In this paper we examine two such applications related to the identification of informative genes and the quantification of regulatory networks. We demonstrate how multiple alternatives can be systematically derived and assess the information content of the proposed solutions.

Recently there has been a paradigm shift in what is considered to be the therapeutic promise of mesenchymal stem cells (MSCs) in diseases of vital organs. Originally, research focused on MSCs as a source of regenerative cells by differentiation of transplanted cells into lost cell types. It is now clear that trophic modulation of inflammation, cell death, fibrosis, and tissue repair are the main mechanisms of MSC therapy. Delivery of growth factors, cytokines, and other signaling molecules secreted by MSCs is often sufficient to obtain the therapeutic effects. In this article, we provide an overview of the current knowledge on trophic mechanisms of MSC therapy in disease models of vital organs. Important issues regarding the optimal delivery methods of MSC therapy are discussed and critical gaps in our knowledge hampering experimental progress and clinical implementation are identified.

Pluripotent embryonic stem cells represent a promising renewable cell source to generate a variety of differentiated cell types including hepatocyte lineage cells, and may ultimately be incorporated into extracorporeal bioartificial liver devices and cell replacement therapies. Recently, we and others have utilized sodium butyrate to directly differentiate hepatocyte-like cells from murine embryonic stem cells cultured in a monolayer configuration. However, to incorporate stem cell technology into clinical and pharmaceutical applications, and hopefully increase the therapeutic potential of these differentiated cells for liver disease treatment, a major challenge remains in sustaining differentiated functions for an extended period of time in their secondary culture environment. In the present work, we have investigated the use of polyacrylamide hydrogels with defined mechanical compliances as a cell culture platform for improving and/or stabilizing functions of these hepatocyte-like cells. Several functional assays, e.g., urea secretion, intracellular albumin content, and albumin secretion, were performed to characterize hepatic functions of cells on polyacrylamide gels with stiffnesses of 5, 46.6, and 230 kPa. In conjunction with the mechanical and cell morphological characterization, we showed that hepatic functions of sodium butyrate differentiated cells were sustained and further enhanced on compliant substrates. This study promises to offer insights into regulating stem cell differentiation via mechanical stimuli, and assist us with designing a variety of dynamic culture systems for applications in tissue and cellular engineering.

Both tissue engineering and biological science will benefit from improved methods to control the morphology, differentiated state, and function of primary cells. In this paper, we show that surface modification of tissue culture polystyrene (TCPS) with chemically derivatized elastin-like polypeptides (ELPs) enables control over the in vitro morphology and liver-specific function of primary rat hepatocytes. The ELP (VPGVG)40 was produced in Escherichia coli and conjugated with polyacrylic acid (PAA) and polyethyleneimine (PEI) using carbodiimide activation chemistry. These conjugates were characterized by transmission Fourier transform infrared (FTIR) spectroscopy, mass spectroscopy, and the ninhydrin assay. We demonstrated that the ELP-polyelectrolyte conjugates profoundly influenced the morphology, aggregation, and differentiated function of primary rat hepatocytes, where hepatocytes plated on the ELP-PAA and ELP-PEI surfaces formed spread and spheroidal morphologies with corresponding low and high liver-specific function, respectively. These materials may have utility as substrata for in vitro studies of hepatocyte biology and tissue engineering applications.

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

Research involving differentiated embryonic stem (ES) cells may revolutionize the study of liver disease, improve the drug discovery process, and assist in the development of stem-cell-based clinical therapies. Generation of ES cell-derived hepatic tissue has benefited from an understanding of the cytokines, growth factors and biochemical compounds that are essential in liver development, and this knowledge has been used to mimic some aspects of embryonic development in vitro. Although great progress has been made in differentiating human ES cells into liver cells, current protocols have not yet produced cells with the phenotype of a mature hepatocyte. There is a significant need to formally establish criteria that would define what constitutes a functional human stem cell-derived hepatocyte. Here, we explore current challenges and future opportunities in development and use of ES cell-derived liver cells. ES-derived hepatocytes could be used to better understand liver biology, begin the process of "personalizing" health care, and to treat some forms of liver disease.

The purpose of this paper is to evaluate the hypothesis that a systems biology approach can be developed such that a set of transcription factors, relevant to burn-induced inflammatory response can be identified and modulated to control the host response. We explore a novel method for identifying coherent and informative expression motifs and we subsequently determine conserved transcription factor binding sites for the sub-sets of co-expressed genes. The responses are rationalized in the context of burn-induced inflammation and the putative transcription factors are rationalized in the context of intervention targets for controlling gene expression.

In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test [Levine JB, Youngs RM, et al. (2007) Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience 145(1):42-55]. In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After 4 weeks of either isolation or group rearing (a period that encompasses post-wearing and early adolescence), rats were killed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post-burn injury. We found that: 1) gross wound healing was significantly impaired in isolation-reared rats compared with group-reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation-reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group-reared rats than for uninjured group-reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation-reared rats during burn injury compared with uninjured isolation-reared rats. Thus, in the mPFC, behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.

Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin. Disclosure of potential conflicts of interest is found at the end of this article.

Burn injury results in immunosuppression; previous work implicated a combination of altered T lymphocyte subpopulations and the elaboration of macrophage-derived mediators. However, the conclusions were based on T cell stimulations in the setting of high-dose polyclonal mitogenic stimuli and a single kinetic time-point. In this study, splenocytes from burned animals were used to examine lymphocyte responses over a multi-day time course following saturating and subsaturating anti-CD3, as well as mixed lymphocyte response (MLR) stimulation. Burn injury resulted in suppressed splenocyte-proliferative responses to high-dose anti-CD3 (2 μg/ml) at all culture time-points (Days 2–5); this inhibition was eliminated by removing macrophages from the splenocyte cultures, by blocking NO production, or by using splenocytes from burned animals congenitally deficient in IFN-γ (IFN-γ−/−). The results are consistent with immunosuppression attributable to burn-induced IFN-γ production, which in turn, drives macrophage NO synthesis (NOS). In MLR cultures, lymphocyte proliferation and IFN-γ production were depressed at later time-points (Days 3–5). APC from burned animals showed no defects as MLR stimulators; T cells from burned animals showed defective, proliferative responses, regardless of the stimulator population. Removing macrophages, adding a NOS inhibitor, or using IFN-γ−/− splenocytes did not restore the MLR response of burned splenocytes. T cells from burned IFN-γ−/− animals also showed depressed proliferation with subsaturating levels of anti-CD3 (0.1 μg/ml); anti-CD-28 augmented the proliferative response. We conclude that burn-induced immunosuppression to authentic antigenic stimulation is related at least in part to defective CD3 signaling pathways and not simply to increased IFN-γ or NO production.

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.

The effective diffusion coefficient of oxygen in pancreatic islets is an important parameter that is needed for the development of models of viability and function of pancreatic islets that are tra...

Background Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial liver (BAL) device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL device for the treatment of FHF. Materials and methods We injected an adenoviral vector encoding human IL-1 receptor antagonist (AdIL-1Ra) into the liver of D-galactosamine (GalN) intoxicated rats via the portal vein. We also transfected primary rat hepatocytes and reversibly immortalized human hepatocytes (TTNT cells) with AdIL-1Ra, and incorporated these transfected hepatocytes into our flat-plate BAL device and evaluated their efficacy in our GalN-induced FHF rat model after 10 h of extracorporeal perfusion. Results Rats injected with AdIL-1Ra showed significant reductions in the plasma levels of hepatic enzymes. Primary rat hepatocytes transfected with AdIL-1Ra secreted IL-1Ra without losing their original synthetic function. Incorporating these cells into the BAL device and testing in a GalN-induced FHF rat model resulted in significant reductions in plasma IL-6 levels and significantly improved animal survival. Incorporating the AdIL-1Ra transfected TTNT cells in the BAL device and testing in the GalN-induced FHF rat model resulted in significantly reduced plasma IL-6 levels, and a trend toward improved survival was seen. Conclusion Hepatocytes producing IL-1Ra are a promising cell source for BAL devices in the treatment of GalN-induced FHF.

The dynamics of gene expression are fundamental to the coordination of cellular responses. Measurement of temporal gene expression patterns is currently limited to destructive low-throughput techniques such as northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and DNA microarrays. We report a scalable experimental platform that combines microfluidic addressability with quantitative live cell imaging of fluorescent protein transcriptional reporters to achieve real-time characterization of gene expression programs in living cells. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors to create a high density matrix of stimulus-response experiments. We demonstrate the approach in the context of hepatic inflammation by acquiring approximately 5000 single-time-point measurements in each automated and unattended experiment. Experiments can be assembled in hours and perform the equivalent of months of conventional experiments. By enabling efficient investigation of dynamic gene expression programs, this technology has the potential to make significant impacts in basic science, drug development, and clinical medicine.

Elastin-like peptides (ELPs) are derivatives of tropoelastin with a unique property that allows them to stay soluble below a certain critical temperature but reversibly form aggregates above that temperature. Since they are derived from tropoelastin, ELPs are biocompatible, non-toxic, and non-immunogenic. The unique properties of ELPs have made them a desirable class of fusion tags used in several biomedical applications including targeted drug delivery and enhancing the half-life of protein drugs. The most significant property of an ELP is that when fused to other proteins, the phase transition property of the ELP is maintained, and the target protein can be purified using the thermally driven property of the ELP. The ELP tag purification process is simple and inexpensive, and involves cycling the protein above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the desired protein, without any need for chromatography. Consequently, using ELPs as a purification tag should be potentially interesting to biopharmaceutical companies who spend a significant percentage of their manufacturing costs on expensive protein purification techniques such as chromatography and filtration. However, ELP tags have not yet been used for commercial protein purification due to some challenges of translating this technique, which has been demonstrated mostly in academic laboratories, to a biotechnology manufacturing environment. The article first reviews the state-of-the-art in protein "ELPylation," and discusses some applications which have benefitted from using ELP as a fusion tag. Then, the article discusses the main advantages of using ELP as a purification tag, and evaluates the remaining hurdles for its implementation in industrial protein production. Biotechnol. Bioeng. 2016;113: 1617-1627. © 2016 Wiley Periodicals, Inc.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

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.

Many tissues in the human body are composed of densely-packed cells and minimal extracellular matrix (ECM). The resulting close cell proximity is important to retain cell viability and function[1]. The ability to generate three-dimensional (3-D) tissues with high-cell packing density and predefined complex structures is of interest to mimic diverse tissues such as liver, cardiac or brain for therapeutics, diagnostics and drug screening applications[2]. The controlled assembly and organization of multi-cellular systems to mimic native tissues is critical to tissue engineering. Many technologies have been proposed to engineer 3-D tissue constructs with control over geometry and spatial cell distribution. Most of these existing technologies are based on assembling cell encapsulating microscale hydrogels[3], scaffold seeding techniques[4] and extrusion/laser-based bioprinting[5]. These scaffold-based techniques are useful for wide applications such as gene delivery[6], bone regeneration[7] and cell differentiation[8]. However, these methods cannot provide physiologically-relevant cell-packing density and control over the cell proximity for bioengineering tissues with low ECM. Scaffold-free tissue engineering focuses on generating highly organized cell constructs, promoting direct cell-cell interactions and interfering with autologous ECM alignment. In this context, spheroid assembly is a recently introduced a strategy of scaffold-free tissue engineering, which provides the most viable method to build larger tissue constructs[9]. Cell spheroids are spherical cell aggregates that provide the highest possible initial cell packing density for bottom-up tissue engineering and are therefore useful as 3-D building blocks for scaffold-free assemblies. Spheroid assembly technologies include spheroid printing[10] via micronozzles and magnetic cell spheroid assembly[11], which requires loading magnetic nanoparticles into cell spheroids. While spheroid printing can produce highly complex tissue constructs, it suffers from long assembly time that prohibits building large, centimeter-scale, constructs. On the other hand, magnetic approach provides rapid assembly for building large structures but it is severely limited by the geometry of tissue structures -dictated by the geometry of the magnet- it can produce and potential adverse effects of magnetic nanoparticles on cell physiology and metabolism[12].

Accessing the venous bloodstream to deliver fluids or obtain a blood sample is the most common clinical routine practiced in the U.S. Practitioners continue to rely on manual venipuncture techniques, but success rates are heavily dependent on clinician skill and patient physiology. In the U.S., failure rates can be as high as 50% in difficult patients, making venipuncture the leading cause of medical injury. To improve the rate of first-stick success, we have developed a portable autonomous venipuncture device that robotically servos a needle into a suitable vein under image guidance. The device operates in real time, combining near-infrared and ultrasound imaging, image analysis, and a 7-degree-of-freedom (DOF) robotic system to perform the venipuncture. The robot consists of a 3-DOF gantry to image the patient's peripheral forearm veins and a miniaturized 4-DOF serial arm to guide the cannula into the selected vein under closed-loop control. In this paper, we present the system architecture of the robot and evaluate the accuracy and precision through tracking, free-space positioning, and in vitro phantom cannulation experiments. The results demonstrate submillimeter accuracy throughout the operating workspace of the manipulator and a high rate of success when cannulating phantom veins in a skin-mimicking tissue model.

Background 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. Aim To evaluate the effect of heparin immobilization on DLM thrombogenicity. Methods 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. Results 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. Conclusions 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.

Immunoassays are widely utilized due to their ability to quantify a vast assortment of biomolecules relevant to biological research and clinical diagnostics. Recently, immunoassay capabilities have been improved by the development of multiplex assays that simultaneously measure multiple analytes in a single sample. However, these assays are hindered by high costs of reagents and relatively large sample requirements. For example, in vitro screening systems currently dedicate individual wells to each time point of interest and this limitation is amplified in screening studies when the investigation of many experimental conditions is necessary; resulting in large volumes for analysis, a correspondingly high cost and a limited temporal experimental design. Microfluidics based immunoassays have been developed in order to overcome these drawbacks. Together, previous studies have demonstrated on-chip assays with either a large dynamic range, high performance sensitivity, and/or the ability to process samples in parallel on a single chip. In this report, we develop a multiplex immunoassay possessing all of these parallel characteristics using commercially available reagents, which allows the analytes of interest to be easily changed. The device presented can measure 6 proteins in 32 samples simultaneously using only 4.2 μL of sample volume. High quality standard curves are generated for all 6 analytes included in the analysis, and spiked samples are quantified throughout the working range of the assay. In addition, we demonstrate a strong correlation (R2 = 0.8999) between in vitro supernatant measurements using our device and those obtained from a bench-top multiplex immunoassay. Finally, we describe cytokine secretion in an in vitro inflammatory hippocampus culture system, establishing proof-of-concept of the ability to use this platform as an in vitro screening tool. The low-volume, multiplexing abilities of the microdevice described in this report could be broadly applied to numerous situations where sample volumes and costs are limiting.

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. peerReviewed

Degenerative skin diseases affect one third of individuals over the age of sixty. Current therapies use various physical and chemical methods to rejuvenate skin; but since the therapies affect many tissue components including cells and extracellular matrix, they may also induce significant side effects, such as scarring. Here we report on a new, non-invasive, non-thermal technique to rejuvenate skin with pulsed electric fields. The fields destroy cells while simultaneously completely preserving the extracellular matrix architecture and releasing multiple growth factors locally that induce new cells and tissue growth. We have identified the specific pulsed electric field parameters in rats that lead to prominent proliferation of the epidermis, formation of microvasculature and secretion of new collagen at treated areas without scarring. Our results suggest that pulsed electric fields can improve skin function and thus can potentially serve as a novel non-invasive skin therapy for multiple degenerative skin diseases.

To evaluate drug and metabolite efficacy on a target organ, it is essential to include metabolic function of hepatocytes, and to evaluate metabolite influence on both hepatocytes and the target of interest. Herein, we have developed a two-chamber microfabricated device separated by a membrane enabling communication between hepatocytes and cancer cells. The microscale environment created enables cell co-culture in a low media-to-cell ratio leading to higher metabolite formation and rapid accumulation, which is lost in traditional plate cultures or other interconnected models due to higher culture volumes. We demonstrate the efficacy of this system by metabolism of tegafur by hepatocytes resulting in cancer cell toxicity.

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for "electric field sinks" in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.