Abhinav Bhushan

In Vitro Platforms for Evaluating Liver Toxicity

The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug-induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information, and their shortcomings have resulted in “silent” hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that (a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells, and endothelial cells; and (b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion, and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.

Long‐term maintenance of a microfluidic 3D human liver sinusoid

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPAs initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies.

Fabrication and Preliminary Results for LiGA Fabricated Nickel Micro Gas Chromatograph Columns

High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-long 50-mum-wide high aspect ratio columns will be the separation component of a handheld gas chromatograph device for detecting semivolatile and volatile compounds. As a first step, 600-mum-deep electrodeposited nickel columns were fabricated. The serpentine columns were sealed and pressure-flow rate characteristics compared with the theoretical values. The response of the sealed columns was studied by running methane gas plugs through uncoated columns with a flame ionization detector at the exit. Negligible flow-induced dispersion was observed in the sealed metal columns. Unretained peak widths of ~15 ms were measured, and the experimental pressure and flow rate distributions matched those predicted by established analytical models within plusmn2.5%. Columns were coated with OV-1 stationary phase using static coating methods. A mixture of four hydrocarbons C6, C8, C10, and C12 was separated in a coated 50 mum by 600 mum by 0.5 m column in less than 2 s at 70 degC

Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans

Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.

Particles and microfluidics merged: perspectives of highly sensitive diagnostic detection

AbstractThere is a growing need for diagnostic technologies that provide laboratories with solutions that improve quality, enhance laboratory system productivity, and provide accurate detection of a broad range of infectious diseases and cancers. Recent advances in micro- and nanoscience and engineering, in particular in the areas of particles and microfluidic technologies, have advanced the “lab-on-a-chip” concept towards the development of a new generation of point-of-care diagnostic devices that could significantly enhance test sensitivity and speed. In this review, we will discuss many of the recent advances in microfluidics and particle technologies with an eye towards merging these two technologies for application in medical diagnostics. Although the potential diagnostic applications are virtually unlimited, the most important applications are foreseen in the areas of biomarker research, cancer diagnosis, and detection of infectious microorganisms. FigureThere is a growing need for diagnostic technologies that provide laboratories with solutions that improve quality, enhance laboratory system productivity, and provide accurate detection of a broad range of infectious diseases and cancers. In this review, we will discuss many of the recent advances in microfluidics and particle technologies with an eye towards merging these two technologies for application in medical diagnostics such as microfluidic device to monitor molecular secretions in real-time as demonstrated in this figure.

Endogenous Levels of Five Fatty Acid Metabolites in Exhaled Breath Condensate to Monitor Asthma by High-Performance Liquid Chromatography: Electrospray Tandem Mass Spectrometry

Abstract-Airway inflammation characterizing asthma and other airway diseases may be monitored through biomarker analysis of exhaled breath condensate (EBC). In an attempt to discover novel EBC biomarkers, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was used to analyze EBC from ten control non-asthmatics and one asthmatic individual for five fatty acid metabolites: 9,12,13-trihydroxyoctadecenoic acid (9,12,13-TriHOME), 9,10,13-TriHOME, 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 12-hydroxyeicosatetraenoic acid (12-HETE), and 12(13)-epoxyoctadecenoic acid (12(13)-EpOME). The method was shown to be sensitive, with an on-column limit of quatitation (LOQ) in the pg range (corresponding to pM concentrations in EBC), and linear over several orders of magnitude for each analyte in the calibrated range. Analysis of EBC spiked with the five fatty acid metabolites was within 81%-119% with only a few exceptions. Endogenous levels in EBC exhibited intraand inter-assay precision of 10%-22%, and 12%-36%, respectively. EBC from the healthy subjects contained average analyte levels between 15 and 180 pM with 12-HETE present above the LOQ in only one of the subjects at a concentration of 240 pM. Exposure of the asthmatic subject to allergen led to increased EBC concentrations of 9,12,13-TriHOME, 9,10,13-TriHOME, 12,13-DiHOME, and 12(13)-EpOME when compared to levels in EBC collected prior to allergen exposure (range =40-510 pM). 12,13-DiHOME was significantly increased (Students t-test, p < 0.05). In conclusion, we have developed a new HPLC-ESI-MS/MS method for the analysis of five fatty acid metabolites in EBC, which are potential biomarkers for asthma monitoring and diagnosis.

Machine learning: A crucial tool for sensor design

Sensors have been widely used for disease diagnosis, environmental quality monitoring, food quality control, industrial process analysis and control, and other related fields. As a key tool for sensor data analysis, machine learning is becoming a core part of novel sensor design. Dividing a complete machine learning process into three steps: data pre-treatment, feature extraction and dimension reduction, and system modeling, this paper provides a review of the methods that are widely used for each step. For each method, the principles and the key issues that affect modeling results are discussed. After reviewing the potential problems in machine learning processes, this paper gives a summary of current algorithms in this field and provides some feasible directions for future studies.

Temperature changes in exhaled breath condensate collection devices affect observed acetone concentrations.

Chemical analysis of exhaled breath condensate (EBC) is an emerging method to non-invasively identify and measure potential biomarkers of disease. Various EBC collection methods have been proposed, each with strengths and weaknesses. Recent evidence in the literature suggests that sample collection methodologies could introduce potential artifacts in biomarker measurements. In this study, we tested the effect of thermal changes during condensate collection on measured EBC chemical concentrations. Using both actively-cooled and passively-cooled devices, we measured distinct differences in the amount of condensate that can be collected over discrete time periods. We also found that concentrations of acetone varied with the thermal profile changes in the collection devices, in apparently identical EBC samples. Together, this evidence suggests that great care should be taken to standardize EBC collection methods, and that small deviations in the thermal properties of the collection devices could contribute to confounding EBC measurement artifacts. This has implications for the design and development of future portable breath analysis systems, especially miniature hand-held devices.

Hybrid integration of injector and detector functions for microchip gas chromatography

Hybrid microchips containing high aspect ratio gas chromatograph (GC) columns with an integrated on-chip split injection and a flame ionization detector were developed. Two different column configurations, spiral and serpentine, both 1 m long by 50 μm wide and 500 μm tall, were fabricated out of electrodeposited nickel. The hybrid chip allowed injection plugs on the order of 1-2 ms, which lowered the height equivalent to theoretical plates (HETP) and allowed a comparison of system level band broadening between the two column configurations. The gas phase band broadening was estimated by measuring the flow characteristics and peak broadening of an unretained compound, and the results were compared with kinetic models. Experimental results show that both spiral and serpentine column layouts had similar flow and band broadening, suggesting that gas phase band broadening may be independent of column layout. The necessity for narrow injection bands for fast micro-chip chromatographic analysis was demonstrated, which emphasized the importance of component integration in designing powerful micro-analytical systems.

Fabrication and Testing of High Aspect Ratio Metal Micro-Gas Chromatograph Columns

Micro gas chromatograph (GC) separation columns were fabricated and tested. Rectangular columns with high aspect ratio have inherent benefits over traditional tubular columns and can easily be integrated into a GC sensor system for rapid chromatographic analysis. The 2 m long, 50 μm wide, 500 μm tall columns were fabricated out of electroplated nickel through deep X-ray lithography using the LIGA technique. Critical fabrication issues including reproducible fabrication of compact footprint, deep columns and sealing of the metal columns were addressed. Experiments were conducted to establish the relationship between column head pressure and flow rate. Methane samples analyzed using hydrogen as a carrier gas on uncoated columns showed very little dispersion suggesting that there was no internal leakage in the columns. The sensor performance data compares well with other micro GC sensor systems.Copyright