An organized 3D in vitro model for peripheral nerve studies

Three-dimensional cell cultures have been proposed to address the limitations in two-dimensional cell cultures (i.e. lack of relevant architectural features) and very few have developed 3D cell culture methods for peripheral nerve studies. Therefore, the present study reports on the development of a 3D in vitro peripheral nerve model using aligned electrospun polycaprolactone fibre scaffolds manufactured under tightly controlled and reproducible conditions with uniform diameters of 1 μm, 5 μm and 8 μm. Fibres were characterized by SEM for diameter, density and alignment properties and formed in to scaffolds for 3D in vitro culture. Three different approaches were adopted using: i) neuronal or primary Schwann cell cultures alone; ii) neuronal and primary Schwann cells in co-culture and iii) isolated dorsal root ganglion cultures, containing both neuronal and Schwann cells, with immunohistochemical and 3D confocal microscopy analysis. Neurite guidance was evident on all fibres diameters with the longest neurites detected on 8 μm fibres when cultured alone. However, co-culture with primary Schwann cells was found to enable neurite formation on all scaffolds. Dorsal root ganglion explants when grown on scaffolds showed both organised aligned neurite guidance and notably the co-localization of Schwann cells with neurites. Neurite lengths of up to 2.50 mm were routinely observed using 1 μm diameter fibres after 10 days and all cultures demonstrated a migrating Schwann cell „front‟ of up to 2.70 mm (1 μm diameter fibres). Thus, a direct relationship was found between fibre diameter, neurite outgrowth and Schwann cell behaviour. Myelin formation was also studied in neuronal/Schwann cell co-cultures either as neuronal cells plus primary Schwann cells or as DRG explants, although no myelin expression was detected. This work therefore supports the use of aligned electrospun PCL microfibres for the development of 3D peripheral nerve models in vitro, with future value in a number of areas including developmental biology, disease studies and the design of devices and scaffolds for peripheral nerve repair.