(233d) Controllable Concentration Gradients By Spatially-Patterned Gene Delivery

The proper function of the nervous system is dependent on a precise and complex tissue architecture, characterized by the organization of multiple cells into structures. Gradients of soluble factors guide this organization during tissue development, and regenerative strategies following injury must recreate this complex architecture to restore function. Here, we investigate patterned gene delivery as a means to organize tissue formation, using an in vitro neuronal co-culture model. Using DNA encoding for diffusible guidance factors (NGF), patterned transfection may lead to localized secretion, and the resulting concentration gradients affect neuronal survival and direct neurite extension. We initially investigated the concentration gradients and subsequent neuronal responses resulting from groups of transfected cells. Accessory cells were transfected in lines of 250 ? 1000 µm widths using a substrate-immobilization strategy. The survival and neurite outgrowth of DRG neurons cultured with transfected accessory cells were measured for the transfection patterns. Concentration gradients resulting from the patterned transfections were predicted with mathematical models. The width of the pattern and transfection efficiency influenced protein production, which subsequently affected the distance outside the pattern in which neuron survival occurred. Neuron survival outside the 250 µm pattern of expression was minimal. For 500 ? 1000 µm wide patterns of expression, the extent of neurite guidance was dependent on the rate of protein production and the distance between a neuron and the pattern of NGF expression. We subsequently investigated the concentration gradients that develop around individual cells, and their guidance of neurite outgrowth. Three transfection profiles, defined by the number of transfected cells and extent of transgene expression, were investigated: 1) low number of transfected cells (<10%) with low extent of expression (10⁵ RLU/mg), 2) low number of transfected cells (<10%) with high extent of expression (10⁸ RLU/mg), and 3) high number of transfected cells (>40%) with high extent of expression (10⁸ RLU/mg). The total length of neurites and the number of sprouting neurons were highest in condition 3. However, the greatest length of primary neurites with minimal branching was achieved with condition 1. The expression level and distance between a neuron and a transfected cell affected neuron survival and neurite length and branching. The combination of expression level and transfected cell spacing may be tuned to maximally promote neurite extension with minimal branching. These patterned gene expression systems provide a platform to investigate cellular responses to concentration gradients, and may be applied for the engineering of tissues with complex architectures.