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There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

Carbon nanotubes (CNTs) are used with increasing frequency in neuroengineering applications. CNT scaffolds are used to transmit electrical stimulation to cultured neurons and to control outgrowth and branching patterns of neurites. CNTs have been reported to disrupt normal neuronal function including alterations in endocytotic capability and inhibition of ion channels. Calcium ion channels regulate numerous neuronal and cellular functions including endo and exocytosis, neurite outgrowth, and gene expression. Strong CNT interactions with neuronal calcium ion channels would have profound biological implications. Here we show that physiological solutions containing CNTs inhibit neuronal voltage-gated calcium ion channels in a dose-dependent and CNT sample-dependent manner with IC50 as low as 1.2 μg/ml. Importantly, we demonstrate that the inhibitory activity does not involve tubular graphene as previously reported, but rather very low concentrations of soluble yttrium released from the nanotube growth catalyst. Cationic yttrium potently inhibits calcium ion channel function with an inhibitory efficacy, IC50, of 0.07 ppm w/w. Because of this potency, unpurified and even some reportedly “purified” CNT samples contain sufficient bioavailable yttrium to inhibit channel function. Our results have important implications for emerging nano-neurotechnology and highlight the critical role that trace components can play in the biological response to complex nanomaterials.