The requirement for improved modeling of cells in culture for study of cell-to-cell interactions has led to an increased focus on three-dimensional (3D) in vitro models. In this study, NG108-15 neural cells and rat sciatic nerve Schwann cells were successfully grown as monocultures and as a coculture on the biocompatible nylon mesh substrates. This has allowed proliferation and interactions between the two cell types to be monitored on the mesh over time. Electrophysiological recordings validated the NG108-15 neural cell differentiation which confirmed the similarity of recorded action potentials in two-dimensional culture environments. A variety of assays have been used, demonstrating the use of nylon as an appropriate substrate for neural cell in vitro studies, and nylon can easily be imaged using inverted microscopy. These studies confirm the suitability of the nylon 3D culture presented for modeling neural cell behavior. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1575-1584, 2018.

INTRODUCTION Peripheral nerve regeneration is a complex challenge that requires suitable nerve guidance systems to bridge the severed ends of 2 nerves back together. Current polymeric conduits on the market provide good cellular growth but are limited by the length of gap defect they can repair, and complete functional recovery is rare. This project focused on creating a three-dimensional (3D) in Vitro spheroidal sprouting assay for peripheral nerve regeneration, as well as producing and testing different polymeric hydrogels as potential scaffold materials for the conduit. METHODS Different concentrations of chitosan, methylcellulose (MC) and sodium alginate were produced, as well as blends of these materials. These hydrogels were seeded with 3D neurospheroids, along with NG108-15 (neuronal) cells and Schwann cells to test their biocompatibility. RESULTS MTT assays showed the mean absorbance of chitosan gels with NG108-15 cells at 24 hr (P < .001) and 72 hr (P > .05) was similar/slightly higher than the negative control. Live-Dead data showed 93.4% of live cells at DIV7 on MC: Ch blends, compared to 72% with chitosan alone. CONCLUSION Overall, both chitosan and MC were nontoxic and biocompatible with NG108-15 and Schwann cells. Blending chitosan with MC improved its chemical and physical properties. The cells formed spheroids that well on a gel; this pseudo-3D structure is excellent for research purposes compared to 2D as it mimics the body's internal environment.