Purpose of review
The capacity of the liver to regenerate and maintain a constant size despite injury is unique. However, the exact mechanisms are not completely clear. Cell transplantation has been proposed as an alternative treatment of liver diseases. Recent progress has been reported on the generation of stem/progenitor cells that may differentiate towards the hepatic lineage. However, it is currently difficult to determine which of the stem/progenitor cell populations are the best for therapy of a given disease.
The limited access to donor human hepatocytes has opened a great interest on the generation of hepatocyte-like cells. Several potential cell sources have been identified. However, general standardization of the methods to evaluate these cells is particularly important for the promise of stem/progenitor-derived hepatocyte-based therapies. Moreover, innovations aimed at improving hepatocyte delivery, survival and engraftment have recently opened the field of organ engineering that may improve the perspective of liver repopulation.
Here we review current evidence reported from the perspective of potential clinical applications of different hepatic cell sources with repopulation capacities and the future perspectives and tools that can facilitate the translation of laboratory work into clinical success.
Hepatocyte transplantation; xenogenic hepatocytes; stem cell-derived hepatocytes; liver tissue engineering
Dendritic cell chemotaxis is an important process involved in the acquisition of adaptive immunity. Despite several studies, our understanding of this process remains limited. One of the reasons for this is the lack of experimental models that give us real-time information on dendritic cell locomotion. Here, using tools in microfluidics, we have fabricated a microdevice that allows us to monitor dendritic cell migration in a chemokine gradient in real time. We successfully observed the migration of dendritic cells derived from a myeloid leukemia cell line (MUTZ-3) in a soluble chemokine (CCL-19) gradient. Our experiments suggest the utility of microdevices in monitoring dendritic cell chemotaxis in real time and getting important information regarding migration speeds and distances previously not available from conventional chemotaxis assays. This kind of data is useful for building mechanistic mathematical models of dendritic cell chemotaxis that may give us novel insights to the process of dendritic cell chemotaxis.
Chemotaxis; dendritic cells; BioMEMs; migration; CCL-19
Mesenchymal stem cells (MSCs) are capable of modulating the immune system and have been used to successfully treat a variety of inflammatory diseases in preclinical studies. Recent evidence has implicated paracrine signaling as the predominant mechanism of MSC therapeutic activity. We have shown in models of inflammatory organ failure that the factors secreted by MSCs are capable of enhancing survival, downregulating inflammation, and promoting endogenous repair programs that lead to the reversal of these diseases. As a marker of disease resolution, we have observed an increase in serum IL-10 when MSC-conditioned medium (MSC-CM) or lysate (MSC-Ly) is administered in vivo. Here we present an in vitro model of IL-10 release from blood cells that recapitulates this in vivo phenomenon. This assay provides a powerful tool in analyzing the potency of MSC-CM and MSC-Ly, as well as characterizing the interaction between MSC-CM and target cells in the blood.
Mesenchymal stem cell; IL-10; Potency assay; Organ injury; Inflammation; Autoimmunity; Transplantation
Deep partial thickness burns are subject to delayed necrosis of initially viable tissues surrounding the primary zone of thermally induced coagulation, which results in an expansion of the burn wound, both in area and depth, within 48 hours postburn. Neutrophil sequestration and activation leading to microvascular damage is thought to mediate this secondary tissue damage. Resolvins, a class of endogenous mediators derived from omega-3 polyunsaturated fatty acids, have been shown to regulate the resolution of inflammation. We hypothesized that exogenous resolvins could mitigate the deleterious impact of the inflammatory response in burn wounds. Using two different mouse burn injury models involving significant partial thickness injuries, we found that a systemically administered single dose of resolvin D2 (RvD2) as low as 25 pg/g bw given within an interval of up to 4 hours postburn effectively prevented thrombosis of the deep dermal vascular network and subsequent dermal necrosis. By preserving the microvascular network, RvD2 enhanced neutrophil access to the dermis, but prevented neutrophil-mediated damage through other anti-inflammatory actions, including inhibition of tumor necrosis factor-α, interleukin-1β, and neutrophil platelet–endothelial cell adhesion molecule-1. In a clinical context, RvD2 may be therapeutically useful by reducing the need for surgical debridement and the area requiring skin grafting.
Initially hailed as the ultimate solution to organ failure, engineering of vascularized tissues such as the liver has stalled because of the need for a well-structured circulatory system that can maintain the cells seeded inside the construct. A new approach has evolved to overcome this obstacle. Whole-organ decellularization is a method that retains most of the native vascular structures of the organ, providing microcirculatory support and structure, which can be anastomosed with the recipient circulation. The technique was first applied to the heart and then adapted for the liver. Several studies have shown that cells can be eliminated, the extracellular matrix and vasculature are reasonably preserved and, after repopulation with hepatocytes, these grafts can perform hepatic functions in vitro and in vivo. Progress is rapidly being made as researchers are addressing several key challenges to whole-organ tissue engineering, such as ensuring correct cell distribution, nonparenchymal cell seeding, blood compatibility, immunological concerns, and the source of cells and matrices.
Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.
Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.
The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system.
Livers from 12 Yorkshire pigs (20–30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly.
Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts.
Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.
donor after cardiac death (DCD); normothermic machine perfusion (NMP); warm ischemia; transplantation; ATP
Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.
Cytokine; Immune response; Immunomodulation; Macrophage; Microencapsulation; Vaccine
The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fiber bioreactor devices in which the cells (separated by a semipermeable polyethersulfone (PES) membrane) can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibers. The cells also preserved their immunophenotype under physiologic fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow fiber bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.
Mesenchymal Stem Cell; Dialysis; Acute Kidney Failure
Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid P. vanderplanki  to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese Hamster Ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4 M trehalose for 4 h at 37 °C, a seven-fold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14 % relative humidity. After desiccation to 2.60 g of water per gram dry weight, a 170 % increase in viability and a 400 % increase in growth (after 7 days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.
Drug-induced liver injury (DILI) limits the development and utilization of numerous therapeutic compounds, and consequently presents major challenges to the pharmaceutical industry and clinical medicine1, 2. Acetaminophen (APAP) containing compounds are among the most frequently prescribed drugs, and also the most common cause of DILI3. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and APAP-induced hepatotoxicity. We report that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation, and death. We identified a small molecule inhibitor of Cx32 as a novel hepatoprotectant that achieves the same result in wildtype mice when coadministered with known hepatotoxic drugs. These findings demonstrate that gap junction inhibition is an effective therapy for limiting DILI, and suggest a novel pharmaceutical strategy to improve drug safety.
Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.
The current state of the art for linear optimization in Flux Balance Analysis has been limited to single objective functions. Since mammalian systems perform various functions, a multiobjective approach is needed when seeking optimal flux distributions in these systems. In most of the available multiobjective optimization methods, there is a lack of understanding of when to use a particular objective, and how to combine and/or prioritize mutually competing objectives to achieve a truly optimal solution. To address these limitations we developed a soft constraints based linear physical programming-based flux balance analysis (LPPFBA) framework to obtain a multiobjective optimal solutions. The developed framework was first applied to compute a set of multiobjective optimal solutions for various pairs of objectives relevant to hepatocyte function (urea secretion, albumin, NADPH, and glutathione syntheses) in bioartificial liver systems. Next, simultaneous analysis of the optimal solutions for three objectives was carried out. Further, this framework was utilized to obtain true optimal conditions to improve the hepatic functions in a simulated bioartificial liver system. The combined quantitative and visualization framework of LPPFBA is applicable to any large-scale metabolic network system, including those derived by genomic analyses.
Bioartificial Liver; Hepatocytes/Linear Physical Programming; Metabolic Networks; Multiobjective Optimization; Pareto Optimality
Biomaterials; cell-material interaction; drug delivery; multifunctional coatings; nanoporous materials
Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 µm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.
hepatocytes; co-culture; layered cell patterning; cellular interactions; fibroblasts
Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.
Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury-models induced by Lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.
anti-inflammation; anti-apoptosis; sepsis; burn; lipopolysaccharide (LPS); systemic inflammatory response syndrome (SIRS); and multiple organ injury
Fatty liver is a significant risk factor for liver transplantation, and accounts for nearly half of the livers rejected from the donor pool. We hypothesized that metabolic preconditioning via ex vivo perfusion of the liver graft can reduce fat content and increase post-transplant survival to an acceptable range. We describe a perfusate medium containing agents that promote the defatting of hepatocytes and explanted livers. Defatting agents were screened on cultured hepatocytes made fatty by pre-incubation with fatty acids. The most effective agents were then used on fatty livers. Fatty livers were isolated from obese Zucker rats and normothermically perfused with medium containing a combination of defatting agents. This combination decreased the intracellular lipid content of cultured hepatocytes by 35% over 24 hours, and of perfused livers by 50% over 3 hours. Metabolite analysis suggests that the defatting cocktail upregulated both lipid oxidation and export. Furthermore, gene expression analysis for several enzymes and transcription factors involved in fatty acid oxidation and triglyceride clearance were elevated. We conclude that a cocktail of defatting agents can be used to rapidly clear excess lipid storage in fatty livers, thus providing a new means to recondition donor livers deemed unacceptable or marginally acceptable for transplantation.
nuclear receptors; normothermic perfusion; hepatocytes; steatosis; liver transplantation
Organotypic brain slice cultures are used for a variety of molecular, electrophysiological, and imaging studies. However, the existing culture methods are difficult or expensive to apply in studies requiring long-term recordings with multielectrode arrays (MEAs). In this work, a novel method to maintain organotypic cultures of rodent hippocampus for several weeks on standard MEAs in an unmodified tissue culture incubator is described. Polydimethylsiloxane (Sylgard) mini-wells were used to stabilize organotypic cultures on glass and MEA surfaces. Hippocampus slices were successfully maintained within PDMS mini-wells for multiple weeks, with preserved pyramidal layer organization, connectivity, and activity. MEAs were used to record the development of spontaneous activity in an organotypic cultures for four weeks. This method is compatible with integration of microchannels into the culture substrate. Microchannels were incorporated into the mini-wells and applied to the guidance of axons originating within the slice, paving the way for studies of axonal sprouting using organotypic slices.
organotypic; hippocampus; multielectrode array; MEA; PDMS; soft lithography; microchannel; axon
Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in-vitro and in-vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In-vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 hours post- injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post SCI indicated that as few as 5×104 encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in-vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.
Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET.
Mitoneet; Autodock; Docking; Fluorescence; Iron–sulfur; Thiazolidinedione
Extending transplant criteria to include livers obtained from donor after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and is therefore perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex-vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N=10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N=6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.
With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer.
Microfluidic droplets; RCA; single-molecule detection; antigen-antibody; tumor markers