Optimal neural differentiation and extension of hybrid neuroblastoma cells (NDC) for nerve-target evaluations using a multifactorial approach.

In vitro models of tissues, such as the cornea, represent systems for modeling cell-to-cell interactions and tissue function. The objective of this study was to develop an optimized nerve differentiation medium to incorporate into a 3D in vitro model to study innervation and cell targeting. A hybrid neuroblastoma cell line (NDC) was examined for its ability to differentiate into neurons, produce neurites, and functionally contact target cells. Neuronal differentiation of NDCs was optimized through a combinatorial approach which involved culturing cells in the presence of various extracellular matrices and soluble factors. A serum-free medium containing nerve growth factor (NGF), dimethyl sulfoxide (DMSO), or dexamethasone resulted in the greatest proportion of NDCs demonstrating a neuronal morphology. Similarly, with supplementation of cyclic AMP (cAMP) or NGF, neurite extension was optimized. Combining these factors generated an optimized differentiation and extension medium, relative to the individual components alone. In co-culture with epithelial cells, NDC neurites generated in the optimized medium formed contacts with epithelial targets and produced substance P. Similarly, NDCs seeded into a collagen matrix produced neurites that projected through the matrix to target epithelial cells, promoted epithelial stratification, and increased the rate of epithelial wound healing. As well, differentiated NDCs could target and alter acetylcholine receptor clustering in mouse C2C12 myotubes, demonstrating synaptic plasticity. Our data supports the use of NDCs, in combination with optimized medium, for generating an innervated in vitro model. Copyright (c) 2009 Elsevier Ltd. All rights reserved.