Electrophysiological measurements in three-dimensional in vivo-mimetic organotypic cell cultures: Preliminary studies with hen embryo brain spheroids

Using three-dimensional artificial tissue constructs shown to offer organotypic functionality, hen embryo brain spheroids were used as a novel electrophysiological paradigm. For the first time, single spontaneous action potentials were recorded from spheroids in culture at day 7 in vitro (DIV) using multi-electrode arrays. At DIV14 'bursting behaviour' was observed. Simple stimulation was found to induce an increase in spiking frequency with an effect that ramped up over DIV7-14. By DIV14, the frequency under stimulation was typically over twice that of the corresponding spontaneous spiking. These results indicate strong self-organizing processes in vitro within the neuronal networks of the three-dimensional spheroid cell cultures. The organotypic in vivo-mimetic nature of the spheroid paradigm was confirmed by electron microscopy that revealed an outer layer of glial cells, a glial limitans, while immunostaining for Neurofilament and Glial Fibrilliary Acidic Protein demonstrated neuronal cells with a centralized neuronal and synaptic distribution. Basic biochemical functionality was also determined and Acetylcholinesterase measured, indicating the activity of acetylcholine receptors. Thus the organotypic hen embryo brain spheroid model may offer a new paradigm in which to explore neuronal networks.