Elishai Ezra

Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction

Significance Microfluidic organ-on-a-chip technology is poised to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Here we demonstrate a sensor-integrated platform permitting real-time tracking of the dynamics of metabolic adaptation to mitochondrial dysfunction. Our approach permits detection of chemical toxicity before any effects on cell or tissue viability can be observed. Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology.

Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection

Viruses lack the basic machinery needed to replicate and therefore must hijack the hosts metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a hosts metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.

Non-dimensional analysis of retinal microaneurysms: critical threshold for treatment

Fluid dynamics play a fundamental role in the development of diabetic retinopathy, one of the leading causes of blindness in the Western world, affecting over 4 million people in the US alone. The disease is defined by microaneurysms, local expansions of capillaries that disturb the hemodynamic forces experienced by the endothelium leading to dysfunction, leakage and edema. Here we present a method to identify microaneurysms with a high risk of leakage based on a critical ratio of microaneurysm to vessel diameter. We derive this non-dimensional parameter from an analytical solution and generalize it using experimentally validated numerical methods. We show that this non-dimensional parameter defines the shear force experienced by endothelial cells, below which endothelial dysfunction is evident in vivo. Our results demonstrate the involvement of vWF in diabetic retinopathy, and explain a perceived disconnect between microaneurysm size and leakage. This method will allow experts to treat microaneurysms poising a high-risk of leakage, prior to edema, minimizing damage and saving vision.

Microprocessor-based integration of microfluidic control for the implementation of automated sensor monitoring and multithreaded optimization algorithms

Microfluidic applications range from combinatorial synthesis to high throughput screening, with platforms integrating analog perfusion components, digitally controlled micro-valves and a range of sensors that demand a variety of communication protocols. Currently, discrete control units are used to regulate and monitor each component, resulting in scattered control interfaces that limit data integration and synchronization. Here, we present a microprocessor-based control unit, utilizing the MS Gadgeteer open framework that integrates all aspects of microfluidics through a high-current electronic circuit that supports and synchronizes digital and analog signals for perfusion components, pressure elements, and arbitrary sensor communication protocols using a plug-and-play interface. The control unit supports an integrated touch screen and TCP/IP interface that provides local and remote control of flow and data acquisition. To establish the ability of our control unit to integrate and synchronize complex microfluidic circuits we developed an equi-pressure combinatorial mixer. We demonstrate the generation of complex perfusion sequences, allowing the automated sampling, washing, and calibrating of an electrochemical lactate sensor continuously monitoring hepatocyte viability following exposure to the pesticide rotenone. Importantly, integration of an optical sensor allowed us to implement automated optimization protocols that require different computational challenges including: prioritized data structures in a genetic algorithm, distributed computational efforts in multiple-hill climbing searches and real-time realization of probabilistic models in simulated annealing. Our system offers a comprehensive solution for establishing optimization protocols and perfusion sequences in complex microfluidic circuits.

Opposing shear-induced forces dominate inertial focusing in curved channels and high Reynolds numbers

Inertial focusing is the migration of particles in fluid toward equilibrium, where current theory predicts that shear-induced and wall-induced lift forces are balanced. First reported in 1961, this Segre-Silberberg effect is particularly useful for microfluidic isolation of cells and particles. Interestingly, recent work demonstrated particle focusing at high Reynolds numbers that cannot be explained by current theory. In this work, we show that non-monotonous velocity profiles, such as those developed in curved channels, create peripheral velocity maxima in which opposing shear-induced forces dominate over wall effects. Similarly, entry effects amplified in high Reynolds flow produce an equivalent trapping mechanism in short, straight channels. This focusing mechanism in the developing flow regime enables a 10-fold miniaturization of inertial focusing devices, while our model corrects long-standing misconceptions about the nature of mechanical forces governing inertial focusing in curved channels.

Frame rate free image velocimetry for microfluidic devices

Here, we introduce Streamline Image Velocimetry, a method to derive fluid velocity fields in fully developed laminar flow from long-exposure images of streamlines. Streamlines confine streamtubes, in which the volumetric flow is constant for incompressible fluid. Using an explicit analytical solution as a boundary condition, velocity fields and emerging properties such as shear force and pressure can be quantified throughout. Numerical and experimental validations show a high correlation between anticipated and measured results, with R2 > 0.91. We report spatial resolution of 2 μm in a flow rate of 0.15 m/s, resolution that can only be achieved with 75 kHz frame rate in traditional particle tracking velocimetry.

Integrated Control of Microfluidics – Application in Fluid Routing, Sensor Synchronization, and Real-Time Feedback Control

Microfluidic applications range from combinatorial chemical synthesis to high-through‐ put screening, with platforms integrating analog perfusion components, digitally control‐ led microvalves, and a range of sensors that demand a variety of communication protocols. A comprehensive solution for microfluidic control has to support an arbitrary combination of microfluidic components and to meet the demand for easy-to-operate sys‐ tem as it arises from the growing community of unspecialized microfluidics users. It should also be an easy to modify and extendable platform, which offer an adequate com‐ putational resources, preferably without a need for a local computer terminal for in‐ creased mobility. Here we will describe several implementation of microfluidics control technologies and propose a microprocessor-based unit that unifies them. Integrated con‐ trol can streamline the generation process of complex perfusion sequences required for sensor-integrated microfluidic platforms that demand iterative operation procedures such as calibration, sensing, data acquisition, and decision making. It also enables the im‐ plementation of intricate optimization protocols, which often require significant compu‐ tational resources. System integration is an imperative developmental milestone for the field of microfluidics, both in terms of the scalability of increasingly complex platforms that still lack standardization, and the incorporation and adoption of emerging technolo‐ gies in biomedical research. Here we describe a modular integration and synchronization of a complex multicomponent microfluidic platform.

One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.