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.
Uroukov
Ivan S
IS
Faculty of Applied Sciences, University of the West of England, Bristol, UK. Ivan.Uroukov@uwe.ac.uk
Ma
Mingwen
M
Bull
Larry
L
Purcell
Wendy M
WM
D000818:Animals; D001921:Brain / Q000166:cytology* / Q000196:embryology* / Q000648:ultrastructure; D002455:Cell Division; D002642:Chick Embryo; D004566:Electrodes; D004594:Electrophysiology; D004625:Embryo, Nonmammalian / Q000166:cytology; D047108:Embryonic Development; D005260:Female; D008855:Microscopy, Electron, Scanning; D009924:Organ Culture Techniques / Q000379:methods; D010063:Ovum