Mainak Das

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.

Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate

This protocol describes a cell culture model to study the differentiation of fetal rat skeletal muscle cells. The model uses serum-free medium, a nonbiological substrate N-1[3(trimethoxysilyl)propyl] diethylenetriamine (DETA) and fabricated microcantilevers to promote the differentiation of dissociated rat myocytes into robust myotubes. In this protocol, we also describe how to characterize the myotubes on the basis of morphology, immunocytochemistry and electrophysiology. Here, four major techniques are employed: fabrication of cantilevers, surface modification of the glass and cantilever substrates with a DETA SAM, a serum-free medium and refined culture techniques. This culture system has potential applications in biocompatibility studies, bioartificial muscle engineering, skeletal muscle differentiation studies and for better understanding of myopathies and neuromuscular disorders. The model can be established in 26–33 d.

Measurement of Contractile Stress Generated by Cultured Rat Muscle on Silicon Cantilevers for Toxin Detection and Muscle Performance Enhancement

Background To date, biological components have been incorporated into MEMS devices to create cell-based sensors and assays, motors and actuators, and pumps. Bio-MEMS technologies present a unique opportunity to study fundamental biological processes at a level unrealized with previous methods. The capability to miniaturize analytical systems enables researchers to perform multiple experiments in parallel and with a high degree of control over experimental variables for high-content screening applications. Methodology/Principal Findings We have demonstrated a biological microelectromechanical system (BioMEMS) based on silicon cantilevers and an AFM detection system for studying the physiology and kinetics of myotubes derived from embryonic rat skeletal muscle. It was shown that it is possible to interrogate and observe muscle behavior in real time, as well as selectively stimulate the contraction of myotubes with the device. Stress generation of the tissue was estimated using a modification of Stoneys equation. Calculated stress values were in excellent agreement with previously published results for cultured myotubes, but not adult skeletal muscle. Other parameters such as time to peak tension (TPT), the time to half relaxation (½RT) were compared to the literature. It was observed that the myotubes grown on the BioMEMS device, while generating stress magnitudes comparable to those previously published, exhibited slower TPT and ½RT values. However, growth in an enhanced media increased these values. From these data it was concluded that the myotubes cultured on the cantilevers were of an embryonic phenotype. The system was also shown to be responsive to the application of a toxin, veratridine. Conclusions/Significance The device demonstrated here will provide a useful foundation for studying various aspects of muscle physiology and behavior in a controlled high-throughput manner as well as be useful for biosensor and drug discovery applications.

A Defined Long-Term In Vitro Tissue Engineered Model of Neuromuscular Junctions

Neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study, a serum-free culture system utilizing defined temporal growth factor application and a non-biological substrate resulted in the formation of robust NMJs. The system resulted in long-term survival of the co-culture and selective expression of neonatal myosin heavy chain, a marker of myotube maturation. NMJ formation was verified by colocalization of dense clusters of acetylcholine receptors visualized using alpha-bungarotoxin and synaptophysin containing vesicles present in motoneuron axonal terminals. This model will find applications in basic NMJ research and tissue engineering applications such as bio-hybrid device development for limb prosthesis and regenerative medicine as well as for high-throughput drug and toxin screening applications.

Electrophysiological and Morphological Characterization of Rat Embryonic Motoneurons in a Defined System

In an attempt to integrate biological components with silicon‐based devices and systems, artificial silane surfaces have been successfully used to grow motoneurons in a defined environment. In this study we characterized the morphology and electrophysiology of purified rat embryonic (E14) motoneurons grown on a self‐assembled monolayer (SAM) of N‐1[3‐(trimethoxysilyl)propyl]diethylenetriamine (DETA) versus that on ornithine/laminin surfaces in serum‐free media. On DETA motoneurons were flat and grew more processes, whereas on ornithine/laminin they tended to aggregate. The membrane time constant, a characteristic associated with electrotonic compactness, was significantly longer for motoneurons grown on DETA. Other electrophysiological parameters were similar for the motoneurons on the different surfaces. This is the first study where purified ventral horn motoneurons were cultured in a completely defined (nonbiological surface, serum‐free) environment.

Cerium oxide nanoparticles protect rodent lungs from hypobaric hypoxia-induced oxidative stress and inflammation

Background Cerium oxide nanoparticles (nanoceria) are effective at quenching reactive oxygen species (ROS) in cell culture and animal models. Although nanoceria reportedly deposit in lungs, their efficacy in conferring lung protection during oxidative stress remains unexplored. Thus, the study evaluated the protective efficacy of nanoceria in rat lung tissue during hypobaric hypoxia. Methods A total of 48 animals were randomly divided into four equal groups (control [C], nanoceria treated [T], hypoxia [H], and nanoceria treated plus hypoxia [T+H]). Animals were injected intraperitoneally with either a dose of 0.5 μg/kg body weight/week of nanoceria (T and T+H groups) or vehicle (C and H groups) for 5 weeks. After the final dose, H and T+H animals were challenged with hypobaric hypoxia, while C and T animals were maintained at normoxia. Lungs were isolated and homogenate was obtained for analysis of ROS, lipid peroxidation, glutathione, protein carbonylation, and 4-hydroxynonenal-adduct formation. Plasma was used for estimating major inflammatory cytokines using enzyme-linked immunosorbent assay. Intact lung tissues were fixed and both transmission electron microscopy and histopathological examinations were carried out separately for detecting internalization of nanoparticles as well as altered lung morphology. Results Spherical nanoceria of 7–10 nm diameter were synthesized using a microemulsion method, and the lung protective efficacy of the nanoceria evaluated during hypobaric hypoxia. With repeated intraperitoneal injections of low micromole concentration, we successfully localized the nanoceria in rodent lung without any inflammatory response. The lung-deposited nanoceria limited ROS formation, lipid peroxidation, and glutathione oxidation, and prevented oxidative protein modifications like nitration and carbonyl formation during hypobaric hypoxia. We also observed reduced lung inflammation in the nanoceria-injected lungs, supporting the anti-inflammatory properties of nanoceria. Conclusion Cumulatively, these results suggest nanoceria deposit in lungs, confer protection by quenching noxious free radicals during hypobaric hypoxia, and do not evoke any inflammatory response.

A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Background Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimers disease. Most therapeutic development studies have focused on toxicity directly at the synapse. Methodology/Principal Findings Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Aβ (1–42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods. Conclusions/Significance The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment.

ADULT RAT SPINAL CORD CULTURE ON AN ORGANOSILANE SURFACE IN A NOVEL SERUM-FREE MEDIUM

SummaryIn this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4–6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.

Skeletal Muscle Tissue Engineering: A Maturation Model Promoting Long Term Survival of Myotubes, Structural Development of the Excitation - Contraction Coupling Apparatus and Neonatal Myosin Heavy Chain Expression

The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.

Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers

The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca(2+) currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinsons. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback.