Investigation of the behavior of serum and plasma in a microfluidics system

There are common problems with adsorption of analytes on the surfaces of microfluidic systems with physiological samples such as blood serum, plasma, and urine. The authors\u27 investigation involves the interaction of serum components with fused-silica surfaces under various flow regimes in microcapillaries. Their focus will include the individual components of serum as well as fresh whole serum. The authors studied the whole serum components in our microfluidic system to uncover the responses of proteins in capillary and microchannel surfaces when influenced by the highly variable serum constituents. They have observed the whole serum with a total protein assay by using the bicinchoninic acid assay in combination with a characterization method, such as SDS polyacrylamide gel electrophoresis, and repeated observations for any change of flow rate in fused-silica capillaries (50 mu m inside diameter) under continuous flow. The authors\u27 preliminary results contradict anecdotal evidence that proteins and other components of serum clog or prevent flow at steady low flow rates

Comparison Of Nmda And Ampa Channel Expression And Function Between Embryonic And Adult Neurons Utilizing Microelectrode Array Systems

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke

In Vitro Modeling Of Nervous System: Engineering Of The Reflex Arc

Neural models are invaluable for understanding the physiology and pathology of the nervous system as well as for developing therapeutic strategies targeting relevant injury and diseases. New developments in the field of stem cells enable great feasibility and potential for generating in vitro models of the nervous system, especially human-based models to study diseases and for drug screening. The reflex arc has been a popular model system for studying neural regulation and circuit modulation. Numerous in vitro models of this system have been generated, among which modeling of the efferent portion of the reflex arc, the connection between motoneurons and skeletal muscles, or the neuromuscular junction (NMJ), has been the central focus. To a lesser extent, the afferent portion, or intrafusal fiber to sensory neuron segment, has also been studied as well as the sensory neuron to motoneuron connections. Furthermore, the integration of interdisciplinary technologies such as surface patterning, microelectrode arrays, and cantilever systems is driving biological NMJ systems more toward in vitro platforms for high content and high throughput capabilities which are suitable for drug screening. To better mimic the in vivo condition, inclusions of other components are also in progress, such as the blood-brain barrier, Bio-MEMs technologies and multi-organ-on-a-chip systems. The concurrent progress in integration of biology and engineering will accelerate the development of these in vitro nervous system models which have an increasing suitability for studying physiology and pathology of the human nervous system as well as for use in drug discovery research

Stem Cell Derived Phenotypic Human Neuromuscular Junction Model For Dose Response Evaluation Of Therapeutics

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle\u27s contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs

Patterned Cardiomyocytes On Microelectrode Arrays As A Functional, High Information Content Drug Screening Platform

Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes. © 2011 Elsevier Ltd

A Computational Metabolic Model Of The Ng108-15 Cell For High Content Drug Screening With Electrophysiological Readout

Computational Systems Modeling could play a significant role in improving and speeding up of the drug development process. By the incorporation of cellular modeling into a High Information Content Drug Screening platform the information content of the pharmacological test could be significantly increased through a deeper understanding of cellular pathways and signaling mechanisms. Unfortunately, many of the cellular signaling pathways in the cells are yet to be explored. Moreover, which is an even larger problem, their integration into a functional signaling network at the whole cell level is almost unknown or untested. Thus, there is an urgent need to develop a data-driven functional whole-cell model which enables the correlation of biochemical and physiological experimental results at the whole cell level with partial information available for the metabolic and signal transduction pathways of the cell. We have built a wholecell model of NG108-15 cells and validated some of the underlying cellular metabolic and signal transduction networks with a series of detailed experiments in order to predict cellular responses to a wide variety of extracellular stimuli. This validated assay system will be an important tool for the identification of cellular changes and activation of signal transduction pathways based on changes of electrophysiological properties and responses of the cell and would have a high impact on drug screening and toxicity evaluation at the cell-system level

Transmission electron microscopy study of the cell–sensor interface

An emerging number of micro- and nanoelectronics-based biosensors have been developed for non-invasive recordings of physiological cellular activity. The interface between the biological system and the electronic devices strongly influences the signal transfer between these systems. Little is known about the nanoscopic structure of the cell–sensor interface that is essential for a detailed interpretation of the recordings. Therefore, we analysed the interface between the sensor surface and attached cells using transmission electron microscopy (TEM). The maximum possible resolution of our TEM study, however, was restricted by the quality of the interface preparation. Therefore, we complemented our studies with imaging ellipsometry

A Computational Metabolic Model of the NG108-15 cell for High Content Drug Screening with Electrophysiological Readout

Computational Systems Modeling could play a significant role in improving and speeding up of the drug development process. By the incorporation of cellular modeling into a High Information Content Drug Screening platform the information content of the pharmacological test could be significantly increased through a deeper understanding of cellular pathways and signaling mechanisms. Unfortunately, many of the cellular signaling pathways in the cells are yet to be explored. Moreover, which is an even larger problem, their integration into a functional signaling network at the whole cell level is almost unknown or untested. Thus, there is an urgent need to develop a data-driven functional whole-cell model which enables the correlation of biochemical and physiological experimental results at the whole cell level with partial information available for the metabolic an