Interfacing micron-sized electrodes with neural tissue could potentially transform the treatment of pathological conditions such as depression, pain, paralysis, and neural trauma. To realize these possibilities, one must first understand neural network dynamics and processing capabilities through long-term neuronal signal recording. Although putative in vitro neural network studies employ planar 2-D culture, they may not accurately represent in vivo cellular and network level functions. In contrast, a 3-D in vitro culture model more closely mimics the microenvironments of in vivo neural networks without the extra confounding variables of in vivo neural tissue.

This work characterized an in vitro 3-D neural co-culture model electrophysiologically via multi electrode arrays (MEAs), and morphologically via immunocytochemistry. Since MEA surface insulation SU-8 2000 can be used in neural micro- and multi- electrode arrays, this investigation first developed techniques to make SU-8 2000 cytocompatible. The in vitro 3-D neural co-culture model was then used to study viability and electrophysiological responses to physical injury as well as drugs known to affect network signaling. 1) SU-8 2000 cytotoxicity to neuronal cultures was linked to both poor adhesive properties and toxic components, such as solvents and photo acid generator elements. Surface treatments of oxygen plasma or parylene coating following optimal combinations of heat and isopropanol sonication showed improvement in SU-8 2000 cytocompatibility. 2) The 3-D neural networks within the 3-D co-cultures maintained considerable process outgrowth and complex 3-D structure. The cultures were viable up to three weeks in vitro with functional synaptic connections and spontaneous electrophysiological activity that was responsive to chemical modulation. This electrophysiological activity was modulated by synaptic inhibition. 3) Injury experiments demonstrated that both shear and compression loading significantly increased acute membrane permeability of cells in a strain rate dependent manner. Cell death correlated with higher membrane permeability, and shear resulted in more death than compression in these 3-D cultures.

While techniques were developed for making a major micro-fabrication material cytocompatible, engineering the 3-D neural co-culture resulted in a more physiologicallyrepresentative neural tissue platform, allowing an increased understanding of structurefunction relationships. Overall, this research established and characterized a neural culture system for the mechanistic study of cell growth, cell-cell and cell-matrix interactions, as well as the responses to chemical or mechanical perturbations. This is the first investigation of the network-level electrophysiological activity of 3-D dissociated cultures. This system can be used to model various pathological states in vitro, testing various reparative drugs;​ cell-, and tissue-engineering based strategies;​ as well as for preanimal and pre-clinical testing of neural implants.