The Effects of Cholera Toxin on Cellular Energy Metabolism

Multianalyte microphysiometry, a real-time instrument for simultaneous measurement of metabolic analytes in a microfluidic environment, was used to explore the effects of cholera toxin (CTx). Upon exposure of CTx to PC-12 cells, anaerobic respiration was triggered, measured as increases in acid and lactate production and a decrease in the oxygen uptake. We believe the responses observed are due to a CTx-induced activation of adenylate cyclase, increasing cAMP production and resulting in a switch to anaerobic respiration. Inhibitors (H-89, brefeldin A) and stimulators (forskolin) of cAMP were employed to modulate the CTx-induced cAMP responses. The results of this study show the utility of multianalyte microphysiometry to quantitatively determine the dynamic metabolic effects of toxins and affected pathways

A non-invasive fluorescence-based oxygen sensor and platform for studying cell responses to metabolic agents in real-time

A fluorescence-based sensor in a transverse flow/stop measurement platform has been developed to determine real-time changes in oxygen consumption rates for cell metabolic studies. The oxygen sensitive fluorophore platinum octaethylporphyrin was embedded in a cellulose acetate matrix and affixed to a fiber optic bundle, which provided for transmission of the excitation and emission wavelengths of the film. The fiber optic bundle was sealed in a sensor head that can be used in standard 24-well plates common to research labs. The utility of the sensor and sensing platform were determined by measuring the changes in oxygen consumption rates of Candida albicans during 90/30 s flow/stop cycles. Exposure of these cells to metabolic antagonists and an enhancer showed the expected decrease and increase in oxygen consumption rates in real time. The applicability of the platform to biological studies is illustrated by determination of synergistic activities between antifungal drugs and fluoride exposure in Candida albicans. The robustness of the fluorophore film is demonstrated by perfusion with different media and analyte conditions in the absence of cells. For stop cycle time intervals less than 1 minute the sensor exhibited a rapid and fairly linear change in fluorescence intensity to changing oxygen concentrations in the measurement chamber. Flow cycle fluorescence intensities were used as a baseline correction for treating the stop cycle fluorescence peaks

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

Microfluidic applications range from combinatorial chemical synthesis to high-throughput screening, with platforms integrating analog perfusion components, digitally controlled 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 system 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 computational resources, preferably without a need for a local computer terminal for increased mobility. Here we will describe several implementation of microfluidics control technologies and propose a microprocessor-based unit that unifies them. Integrated control 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 implementation of intricate optimization protocols, which often require significant computational 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 technologies in biomedical research. Here we describe a modular integration and synchronization of a complex multicomponent microfluidic platform

Multi-Sensor Arrays for Online Monitoring of Cell Dynamics in in vitro Studies with Choroid Plexus Epithelial Cells

Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events—in this case in choroid plexus functions, however with applicability to other cell types as well

Multi-Sensor Arrays for Online Monitoring of Cell Dynamics in in vitro Studies with Choroid Plexus Epithelial Cells

Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events—in this case in choroid plexus functions, however with applicability to other cell types as well

Botulinum Toxin Suppression of CNS Network Activity In Vitro

The botulinum toxins are potent agents which disrupt synaptic transmission. While the standard method for BoNT detection and quantification is based on the mouse lethality assay, we have examined whether alterations in cultured neuronal network activity can be used to detect the functional effects of BoNT. Murine spinal cord and frontal cortex networks cultured on substrate integrated microelectrode arrays allowed monitoring of spontaneous spike and burst activity with exposure to BoNT serotype A (BoNT-A). Exposure to BoNT-A inhibited spike activity in cultured neuronal networks where, after a delay due to toxin internalization, the rate of activity loss depended on toxin concentration. Over a 30 hr exposure to BoNT-A, the minimum concentration detected was 2 ng/mL, a level consistent with mouse lethality studies. A small proportion of spinal cord networks, but not frontal cortex networks, showed a transient increase in spike and burst activity with exposure to BoNT-A, an effect likely due to preferential inhibition of inhibitory synapses expressed in this tissue. Lastly, prior exposure to human-derived antisera containing neutralizing antibodies prevented BoNT-A induced inhibition of network spike activity. These observations suggest that the extracellular recording from cultured neuronal networks can be used to detect and quantify functional BoNT effects

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

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