Long-term electromagnetic exposure of developing neuronal networks: A flexible experimental setup.

Neuronal networks in vitro are considered one of the most promising targets of research to assess potential electromagnetic field induced effects on neuronal functionality. A few exposure studies revealed there is currently no evidence of any adverse health effects caused by weak electromagnetic fields. Nevertheless, some published results are inconsistent. Particularly, doubts have been raised regarding possible athermal biological effects in the young brain during neuronal development. Therefore, we developed and characterized a flexible experimental setup based on a transverse electromagnetic waveguide, allowing controlled, reproducible exposure of developing neuronal networks in vitro. Measurement of S-parameters confirmed very good performance of the Stripline in the band of 800-1000 MHz. Simulations suggested a flexible positioning of cell culture dishes throughout a large exposure area, as specific absorption rate values were quite independent of their position (361.7 ± 11.4 mW/kg) at 1 W, 900 MHz. During exposure, thermal drift inside cellular medium did not exceed 0.1 K. Embryonic rat cortical neurons were cultivated on microelectrode array chips to non-invasively assess electrophysiological properties of electrogenic networks. Measurements were taken for several weeks, which attest to the experimental setup being a reliable system for long-term studies on developing neuronal tissue.

Growing neuronal islands on multi-electrode arrays using an accurate positioning-µCP device.

BACKGROUND: Multi-electrode arrays (MEAs) allow non-invasive multi-unit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEA's surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging. NEW METHOD: In this paper, we present a novel approach utilizing micro-contact printing (µCP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-µCP (accurate positioning micro-contact printing). COMPARISON WITH EXISTING METHODS: Other approaches presented in the literature using µCP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning. CONCLUSION: Here we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.

A novel automated spike sorting algorithm with adaptable feature extraction.

To study the electrophysiological properties of neuronal networks, in vitro studies based on microelectrode arrays have become a viable tool for analysis. Although in constant progress, a challenging task still remains in this area: the development of an efficient spike sorting algorithm that allows an accurate signal analysis at the single-cell level. Most sorting algorithms currently available only extract a specific feature type, such as the principal components or Wavelet coefficients of the measured spike signals in order to separate different spike shapes generated by different neurons. However, due to the great variety in the obtained spike shapes, the derivation of an optimal feature set is still a very complex issue that current algorithms struggle with. To address this problem, we propose a novel algorithm that (i) extracts a variety of geometric, Wavelet and principal component-based features and (ii) automatically derives a feature subset, most suitable for sorting an individual set of spike signals. Thus, there is a new approach that evaluates the probability distribution of the obtained spike features and consequently determines the candidates most suitable for the actual spike sorting. These candidates can be formed into an individually adjusted set of spike features, allowing a separation of the various shapes present in the obtained neuronal signal by a subsequent expectation maximisation clustering algorithm. Test results with simulated data files and data obtained from chick embryonic neurons cultured on microelectrode arrays showed an excellent classification result, indicating the superior performance of the described algorithm approach.

Electromagnetic exposure of scaffold-free three-dimensional cell culture systems.

In recent years, a number of in vitro studies have reported on the possible athermal effects of electromagnetic exposure on biological tissue. Typically, this kind of study is performed on monolayers of primary cells or cell lines. However, two-dimensional cell layer systems lack physiological relevance since cells in vivo are organized in a three-dimensional (3D) architecture. In monolayer studies, cell-cell and cell-ECM interactions obviously differ from live tissue and scale-ups of experimental results to in vivo systems should be considered carefully. To overcome this problem, we used a scaffold-free 3D cell culture system, suitable for the exploration of electrophysiological effects due to electromagnetic fields (EMF) at 900 MHz. Dissociated cardiac myocytes were reaggregated into cellular spheres by constant rotation, and non-invasive extracellular recordings of these so-called spheroids were performed with microelectrode arrays (MEA). In this study, 3D cell culture systems were exposed to pulsed EMFs in a stripline setup. We found that inhomogeneities in the EMF due to electrodes and conducting lines of the MEA chip had only a minor influence on the field distribution in the spheroid if the exposure parameters were chosen carefully.