Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Molecular self-assembly of two-terminal, voltammetric microsensors with internal references.

Self-assembly of a ferrocenyl thiol and a quinone thiol onto Au microelectrodes forms the basis for a new microsensor concept: a two-terminal, voltammetric microsensor with reference and sensor functions on the same electrode. The detection is based on measurement of the potential difference of current peaks for oxidation and reduction of the reference (ferrocene) and indicator (quinone) in aqueous electrolyte in a two-terminal, linear sweep voltammogram in which a counterelectrode of relatively large surface area is used. The quinone has a half-wave potential, E((1/2)), that is pH-sensitive and can be used as a pH indicator; the ferrocene center has an E(1/2) that is a pH-insensitive reference. The key advantages are that such sensors require no separate reference electrode and function as long as current peaks can be located for reference and indicator molecules.

Orthogonal self-assembled monolayers: alkanethiols on gold and alkane carboxylic acids on alumina.

This work demonstrates the practicality of forming two self-assembled monolayers (SAMs), independently but simultaneously, by adsorption of two different adsorbates from a common solution onto a substrate exposing two different materials at its surface. The experimental procedure and the degree of independence achieved in the resulting SAMs are illustrated by examination of monolayers obtained by adsorption of alkanethiols on gold and alkane carboxylic acids on alumina. This procedure provides a method for modifying the surface characteristics of microlithographically generated patterns and offers a versatile technique for controlling solid-vapor and solid-liquid interfacial properties in systems having patterns with dimensions of the order of 1 micrometer.

Embryonic motoneuron-skeletal muscle co-culture in a defined system.

This paper describes a significant biotechnological advancement by creating a minimalist serum-free defined system to co-culture rat mammalian nerve and muscle cells in order to form functional neuromuscular junctions. To date, all the known in vitro nerve and muscle co-culture models use serum containing media; and while functional neuromuscular junctions (NMJ) are described, they failed to detail or quantify the minimum factors needed to recreate the NMJ in vitro. In this work, we demonstrate the development of a defined motoneuron and muscle co-culture system resulting in the formation of NMJs including: 1) a new culture technique, 2) a novel serum-free medium formulation and 3) a synthetic self-assembled monolayer (SAM) substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). We characterized the culture by morphology, immunocytochemistry, electrophysiology and videography. This model system provides a better understanding of the minimal growth factor and substrate interactions necessary for NMJ formation and provides a basic system that can be utilized for nerve-muscle tissue engineering, regenerative medicine and development of limb prosthetics.

Functional analysis of human intrafusal fiber innervation by human γ-motoneurons.

Investigation of neuromuscular deficits and diseases such as SMA, as well as for next generation prosthetics, utilizing in vitro phenotypic models would benefit from the development of a functional neuromuscular reflex arc. The neuromuscular reflex arc is the system that integrates the proprioceptive information for muscle length and activity (sensory afferent), to modify motoneuron output to achieve graded muscle contraction (actuation efferent). The sensory portion of the arc is composed of proprioceptive sensory neurons and the muscle spindle, which is embedded in the muscle tissue and composed of intrafusal fibers. The gamma motoneurons (γ-MNs) that innervate these fibers regulate the intrafusal fiber's stretch so that they retain proper tension and sensitivity during muscle contraction or relaxation. This mechanism is in place to maintain the sensitivity of proprioception during dynamic muscle activity and to prevent muscular damage. In this study, a co-culture system was developed for innervation of intrafusal fibers by human γ-MNs and demonstrated by morphological and immunocytochemical analysis, then validated by functional electrophysiological evaluation. This human-based fusimotor model and its incorporation into the reflex arc allows for a more accurate recapitulation of neuromuscular function for applications in disease investigations, drug discovery, prosthetic design and neuropathic pain investigations.

Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods.

Inkjet printing of viable mammalian cells.

The purpose of this study was to explore the use of a commercial thermal printer to deposit Chinese Hamster Ovary (CHO) and embryonic motoneuron cells into pre-defined patterns. These experiments were undertaken to verify the biocompatibility of thermal inkjet printing of mammalian cells and the ability to assemble them into viable constructs. Using a modified Hewlett Packard (HP) 550C computer printer and an HP 51626a ink cartridge, CHO cells and rat embryonic motoneurons were suspended separately in a concentrated phosphate buffered saline solution (3 x). The cells were subsequently printed as a kind of "ink" onto several "bio-papers" made from soy agar and collagen gel. The appearance of the CHO cells and motoneurons on the bio-papers indicated an healthy cell morphology. Furthermore, the analyses of the CHO cell viability showed that less than 8% of the cells were lysed during printing. These data indicate that mammalian cells can be effectively delivered by a modified thermal inkjet printer onto biological substrates and that they retain their ability to function. The computer-aided inkjet printing of viable mammalian cells holds potential for creating living tissue analogs, and may eventually lead to the construction of engineered human organs.

Inkjet printing for high-throughput cell patterning.

The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.

Investigation of the factors necessary for growth of hippocampal neurons in a defined system.

We have developed an in vitro system that combines the use of a defined medium with a chemically defined surface for the differentiation of embryonic rat hippocampal neurons. Cells were grown on silica substrates modified with two chemically distinct molecules: poly-D-lysine and an amine-containing organosilane. Cells were dissociated by mechanical or enzymatic methods and grown in serum-containing versus serum-free medium on these surfaces. Our results demonstrate that optimal survival and growth in serum-free medium occurs on the artificial surfaces. X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces both before and after cell cultures. In addition, surface properties such as elemental composition, the initial thickness of the substrate material, and the thickness of material deposited during the course of cell culture were quantified after cell removal. Taken together, the results from the cell culture and surface analysis demonstrate that the media, proteins deposited from the media onto the surface, surface composition, and properties intrinsic to neuronal membranes all interact in a complex fashion to determine whether or not the cells will adhere and survive in culture. In particular, the role of material deposited from the medium onto the culture substratum may be more important than have been previously appreciated. This system allow for the study of neuronal differentiation in a well-defined environment.

Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons.

High resolution substrates, created using patterned self-assembled monolayers, are shown to direct axonal and dendritic process extension at the level of a single hippocampal neuron. Axons and dendrites were identified using morphological characteristics and immunocytochemical markers. Patterns were formed on glass coverslips from a co-planar monolayer of cell adhesive aminosilanes and non-adhesive fluorinated silanes. On patterned surfaces, the percentage of the total number of cells attached to the 0.71 mm2 substrate field with compliance to the 25-micron diameter 'somal adhesion site' reached 41 +/- 7% (mean +/- S.D., 428 cells counted). A total of 76 +/- 11% of cells that adhered to a somal attachment site developed a lone process > or = 100 microns oriented in the direction of the continuous aminosilane pathway which was shown to express axonal markers. Cells on either the fluorinated silane, which is non-permissive for neurite outgrowth, or localized on an aminosilane region only 5 microns wide failed to extend major processes. This approach is amenable to a variety of industry standard fabrication techniques and may be used to study the role of fine scale spatial cues in neuronal development and synapse formation.

Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.

We have studied the modulation of hippocampal neuron morphological development in vitro using surfaces derivatized with aminosilane self-assembled monolayers (SAMs). The efficacies of model SAMs, alone, or in combination with adsorbed heparan sulfate glycosaminoglycan (HS), are related to the physical and chemical properties of the surfaces. These properties are determined using X-ray photoelectron spectroscopy (XPS), optical ellipsometry, and wettability measurements. The ability of surfaces to promote somal adhesion and the maintenance of discrete neurites appears to be sensitive to the density and accessibility of positively charged amine or amide groups, and has less of an apparent relationship to the surface density of uncharged amines. Aromatic ring-containing aminosilanes are ineffective in promoting neuron growth, while adsorbed HS augments the neurite-promoting capacity of one marginally adhesive SAM. These results are relevant to an improved understanding of the 'non-specific' contributions of the substrate in affecting neuronal development and the rational design of model surface coatings for neuronal culture.

Neuronal and glial epitopes and transmitter-synthesizing enzymes appear in parallel with membrane excitability during neuroblastoma x glioma hybrid differentiation.

The membrane excitability and the presence of neural proteins, including neuronal and glial markers and neurotransmitter-synthesizing enzymes, were examined in parallel while the NG108-15 cell line was maintained in a serum-free medium. Whole-cell recordings in voltage-clamp or current-clamp configurations were used to evaluate the membrane excitability, and immunostaining was done with a panel of well-characterized antibodies against NSE, NF150, S-100 beta, GFAP, ChAT and TH. Culture for 4 to 10 days led to a striking rise in neurite outgrowth, electrical excitability and expression of neural proteins in type I neuron-like cells, which were of both neuronal and glial character, and expressed both cholinergic and adrenergic traits. After about 2 weeks, type II cells which lack neurite processes began to emerge. The type II cells proliferated, as revealed by BrdU uptake, and gradually overgrew differentiated cell types. They exhibited little or no membrane excitability and absence of immunoreactivity for the neuronal and glial specific proteins tested. These measurements indicate that the presence of these neural proteins at crucial stages of membrane excitability development is an important characteristics of NG108-15 cell differentiation, providing insights into the neural development and the reversible nature of neoplasia in the nervous system.

Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system.

The ability to accurately measure skeletal muscle functional performance at the single-cell level would be advantageous for exercise physiology studies and disease modeling applications. To that end, this study characterizes the functional response of individual skeletal muscle myotubes derived from adult rodent tissue to creatine treatment and chronic exercise. The observed improvements to functional performance in response to these treatments appear to correlate with alterations in hypertrophic and mitochondrial biogenesis pathways, supporting previously published in vivo and in vitro data, which highlights the role of these pathways in augmenting skeletal muscle output. The developed system represents a multiplexed functional in vitro assay capable of long-term assessment of contractile cellular outputs in real-time that is compatible with concomitant molecular biology analysis. Adoption of this system in drug toxicity and efficacy studies would improve understanding of compound activity on physical cellular outputs and provide more streamlined and predictive data for future preclinical analyses.

A multiplexed chip-based assay system for investigating the functional development of human skeletal myotubes in vitro.

This report details the development of a non-invasive in vitro assay system for investigating the functional maturation and performance of human skeletal myotubes. Data is presented demonstrating the survival and differentiation of human myotubes on microscale silicon cantilevers in a defined, serum-free system. These cultures can be stimulated electrically and the resulting contraction quantified using modified atomic force microscopy technology. This system provides a higher degree of sensitivity for investigating contractile waveforms than video-based analysis, and represents the first system capable of measuring the contractile activity of individual human muscle myotubes in a reliable, high-throughput and non-invasive manner. The development of such a technique is critical for the advancement of body-on-a-chip platforms toward application in pre-clinical drug development screens.

A functional system for high-content screening of neuromuscular junctions in vitro.

High-content phenotypic screening systems are the logical extension of the current efficient, yet low information content, pre-clinical screens for drug discovery. A physiologically accurate in vitro neuromuscular junction (NMJ) screening system would therefore be of tremendous benefit to the study of peripheral neuropathies as well as for basic and applied neuromuscular research. To date, no fully-defined, selective assay system has been developed which would allow investigators to determine the functional output of cultured muscle fibers (myotubes) when stimulated via the NMJ in real time for both acute and chronic applications. Here we present the development of such a phenotypic screening model, along with evidence of NMJ formation and motoneuron initiated neuromuscular transmission in an automated system. Myotubes assembled on silicon cantilevers allowed for measurement of substrate deflection in response to contraction and provided the basis for monitoring the effect of controlled motoneuron stimulation on the contractile behavior. The effect was blocked by treatment with D-tubocurarine, confirming NMJ functionality in this highly multiplexed assay system.

Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.

Rigorous analysis of muscle function in in vitro systems is needed for both acute and chronic biomedical applications. Forces generated by skeletal myotubes on bio-microelectromechanical cantilevers were calculated using a modified version of Stoney's thin-film equation and finite element analysis (FEA), then analyzed for regression to physical parameters. The Stoney's equation results closely matched the more intensive FEA and the force correlated to cross-sectional area (CSA). Normalizing force to measured CSA significantly improved the statistical sensitivity and now allows for close comparison of in vitro data to in vivo measurements for applications in exercise physiology, robotics, and modeling neuromuscular diseases.

An optimization-based study of equivalent circuit models for representing recordings at the neuron-electrode interface.

Extracellular neuroelectronic interfacing is an emerging field with important applications in the fields of neural prosthetics, biological computation, and biosensors. Traditionally, neuron-electrode interfaces have been modeled as linear point or area contact equivalent circuits but it is now being increasingly realized that such models cannot explain the shapes and magnitudes of the observed extracellular signals. Here, results were compared and contrasted from an unprecedented optimization-based study of the point contact models for an extracellular "on-cell" neuron-patch electrode and a planar neuron-microelectrode interface. Concurrent electrophysiological recordings from a single neuron simultaneously interfaced to three distinct electrodes (intracellular, "on-cell" patch, and planar microelectrode) allowed novel insights into the mechanism of signal transduction at the neuron-electrode interface. After a systematic isolation of the nonlinear neuronal contribution to the extracellular signal, a consistent underestimation of the simulated suprathreshold extracellular signals compared to the experimentally recorded signals was observed. This conclusively demonstrated that the dynamics of the interfacial medium contribute nonlinearly to the process of signal transduction at the neuron-electrode interface. Further, an examination of the optimized model parameters for the experimental extracellular recordings from sub- and suprathreshold stimulations of the neuron-electrode junctions revealed that ionic transport at the "on-cell" neuron-patch electrode is dominated by diffusion whereas at the neuron-microelectrode interface the electric double layer (EDL) effects dominate. Based on this study, the limitations of the equivalent circuit models in their failure to account for the nonlinear EDL and ionic electrodiffusion effects occurring during signal transduction at the neuron-electrode interfaces are discussed.

Nanoscale Nonlinear dynamic characterization of the neuron-electrode junction.

Extracellular recordings from neurons using microelectrode and field effect transistor arrays suffer from many problems including low signal to noise ratio, signal attenuation due to counter-ion diffusion from the bulk extracellular medium and a modification of the shape of the cell-generated potentials due to the presence of a highly dispersive dielectric medium in the cell-electrode cleft. Attempts to date to study the neuron-electrode interface have focused on point or area contact linear-equivalent-circuit models. We present here the results obtained from a 'data-true' nonlinear dynamic characterization of the neuron-electrode junction using Volterra-Wiener modeling. For the characterization, NG108-15 cells were cultured on microelectrode arrays and stimulated with broadband Gaussian white noise under voltage clamp mode. A Volterra-Wiener model was then estimated using the input signal and the extracellular signal recorded on the microelectrode. The existence of the second order Wiener kernel confirmed that the recorded extracellular signal had a nonlinear component. The verification of the estimated model was carried out by employing the intracellular action potential as an input to the Volterra-Wiener model and comparing the predicted extracellular response with the corresponding extracellular signal recorded on the microelectrode. We believe that a 'data-true' Volterra-Wiener model of the neuron-electrode junction shall not only facilitate a direct insight into the physicochemical processes taking place at the interface during signal transduction but will also allow one to evolve strategies for engineering the neuron-electrode interface using surface chemical modification of the microelectrodes.

Spatially controlled adhesion, spreading, and differentiation of endothelial cells on self-assembled molecular monolayers.

Chemically modified glass substrates were used to demonstrate differential adhesion, growth, and differentiation of endothelial cells. Endothelial cells were examined for adhesion and growth on glass, glass treated with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA), or EDA with a subsequent treatment with physically adsorbed extracellular matrix components human fibronectin and heparin sulfate. EDA and EDA/human fibronectin showed similar abilities to support adhesion, spreading, and proliferation of endothelial cells. In contrast, heparin sulfate inhibited endothelial cell adhesion to EDA. Differentiation of endothelial cells resulting in precapillary cord formation was triggered by addition of basic fibroblast growth factor (bFGF). On EDA and EDA/human fibronectin bFGF causes confluent endothelial cell monolayers to differentiate and form cords, which resulted in a large-scale spatial redistribution of cells on the surface. Formation of organized neovascular assemblies was demonstrated on coplanar molecular patterns of EDA and a nonadhesive perfluorinated alkylsilane (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchloros ilane (13F). Endothelial cells preferentially adhered to the EDA lines and after 24-48 hr, microfilaments aligned with the long axes of the patterned EDA region. Finally, endothelial cells that became confluent within the confines of the EDA region (bound by the nonadhesive, 13F domains) were observed to differentiate into neovascular cords in long-term culture (7-10 days) with bFGF.

Fabrication of surfaces resistant to protein adsorption and application to two-dimensional protein patterning.

Proteins were attached in defined geometric patterns on a surface. A prerequisite to making a pattern of proteins is generation of surfaces resistant to nonspecific protein adsorption. This was accomplished via oxidation of the thiol terminus of an organosilane self-assembled monolayer film by deep ultraviolet (DUV) irradiation. The resultant surface exhibited marked resistance to protein adsorption. Using a mask to protect regions of the silanized surface from irradiation, proteins were selectively adsorbed or attached via covalent linkage at locations protected from the DUV light. Antibodies immobilized in patterns using this procedure retained their antigen-binding capability. Thus chemistry and DUV lithography were combined to create patterns of active biomolecules which could be used in the microfabrication of electronic devices and biosensors.