Search

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a non-biological, cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle-nerve co-culture. Immunocytochemical evidence of nerve-muscle co-culture has also been documented. Applications for this novel culture system include: biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders and skeletal muscle tissue engineering.

The ability to culture functional adult mammalian spinal-cord neurons represents an important step in the understanding and treatment of a spectrum of neurological disorders including spinal cord injury. Previously, the limited functional recovery of these cells, as characterized by a diminished ability to initiate action potentials and to exhibit repetitive firing patterns, has arisen as a major impediment to their physiological relevance. In this report we demonstrate that single temporal doses of the neurotransmitters serotonin, glutamate (N-acetyl-DL-glutamic acid) and acetylcholine-chloride leads to the full electrophysiological functional recovery of adult mammalian spinal-cord neurons, when they are cultured under defined serum-free conditions. Approximately 60% of the neurons treated regained their electrophysiological signature, often firing single, double and, most importantly, multiple action potentials.

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.

This paper describes the evaluation of the auto-catalytic anti-oxidant behavior and biocompatibility of cerium oxide nanoparticles for applications in spinal cord repair and other diseases of the central nervous system. The application of a single dose of nano-ceria at a nano-molar concentration is biocompatible, regenerative and provides a significant neuroprotective effect on adult rat spinal cord neurons. Retention of neuronal function is demonstrated from electrophysiological recordings and the possibility of its application to prevent ischemic insult is suggested from an oxidative injury assay. A mechanism is proposed to explain the auto-catalytic properties of these nanoparticles.

SUMMARY In 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, cardiotrophin1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet1 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.

A very small population of Choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). Using this method a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in culture. Interestingly, most of the glutamatergic neurons which expressed glutamate vesicular transporter (VGLUT1) also co-expressed choline acetyltransferase (ChAT) proteins. On the contrary, when the cultures were double stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.

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.

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.

We have developed a method to organize cells in dissociated cultures using engineered chemical clues on a culture surface and determined their connectivity patterns. Although almost all elements of the synaptic transmission machinery can be studied separately in single cell models in dissociated cultures, the complex physiological interactions between these elements are usually lost. Thus, factors affecting synaptic transmission are generally studied in organotypic cultures, brain slices, or in vivo where the cellular architecture generally remains intact. However, by utilizing engineered neuronal networks complex phenomenon such as synaptic transmission or synaptic plasticity can be studied in a simple, functional, cell culture-based system. We have utilized self-assembled monolayers and photolithography to create the surface templates. Embryonic hippocampal cells, plated on the resultant patterns in serum-free medium, followed the surface clues and formed the engineered neuronal networks. Basic whole-cell patch-clamp electrophysiology was applied to characterize the synaptic connectivity in these engineered two-cell networks. The same technology has been used to pattern other cell types such as cardiomyocytes or skeletal muscle fibers.

BACKGROUND Soluble oligomers of amyloid beta (Abeta) are considered to be one of the major contributing factors to the development of Alzheimer's 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 Abeta 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 Abeta (1-42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Abeta 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.