Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.