Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: A comparison of neuronal and myotube extracellular action potentials

Abstract

Microelectrode array (MEA) technology holds tremendous potential in the fields of bio-detection, lab-on-a-chip applications, and tissue engineering by facilitating non-invasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

“…A small fraction of recorded individual gMμEs comprised FPs that were most likely generated by a number of adjacent myotubes. It should be noted that the average amplitudes of the FPs recorded by the gMμE-MEAs were significantly larger than those reported in two earlier publications by Langhammer et al 2122,. using substrate integrated planar electrode based MEA with a surface area of 314  μm 2  2122.…”

“…In contrast to the extensive use of substrate integrated MEA devices in neuroscience and cardiology research only two published papers have examined the use of MEA for in vitro skeletal myotube research. In these studies Langhammer et al 2122. demonstrated that substrate integrated planar MEA, with a surface area of 314 μm 2 , record multiphasic spontaneous FPs from rat cultured myotubes with an average amplitude of 100 μV.…”

“…Analysis of the recorded FPs suggested that even when grown on unstructured substrate myotubes undergo the essential developmental cascades to become electrophysiologically independent of each other. The electrical independence of myotubes is a critical feature that enables skeletal muscles to generate graded and controlable generation and contraction dynamics2122.…”

“…One plausible reason why MEA technology has not been adopted for skeletal muscle research and diagnostics is because progress in the culturing technologies of skeletal myotubes and NMJ from rodents and human is relatively recent151617181920. To date, only two original studies have been published on the use of substrate integrated planar MEA to electrophysiologically record from cultured skeletal myotubes2122.…”

‘Body-on-a-Chip’ Technology and Supporting Microfluidics

“…Measurement of the electrophysiological properties of cell types such as neurons, cardiomyocytes and skeletal muscle permits direct functional assessment of these cells in real time. [25][26][27] Electrical impedance measurements are attractive as a means to measure cell viability in a continuous and non-invasive manner, 28 and the use of microelectrodes could also be implemented for measurement of a wide variety of relevant metabolites such as lactate. 29 In addition, application of technologies designed to measure physical displacement, such as exible posts 30 or microscale cantilever arrays, 31 could facilitate measurement of force generation in cardiac, skeletal and smooth muscle tissues, providing analytical assessment of physical maturation within multi-organ models.…”