Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB275

Poster Communications

A novel aligned dorsal root ganglion explant culture system using 3D printed substrates

L. Alvey1, K. Turner1, J. F. Jones1, M. Pickering1

1. School of Medicine, University College Dublin, Glasthule, Ireland.

Directing axonal growth to targets is challenging and expensive, including techniques that utilise microfluidic devices and nanofiber scaffolds. 3D printing is an inexpensive, efficient, versatile and rapidly evolving method of building 3D constructs, and may provide opportunities to build systems to study directed neural migration and nerve regeneration. Dorsal root ganglion (DRG) explants are used to elucidate the migration of sensory neurons and their attendant glia, Schwann cells. If cultured on flat surfaces, nerve fibres grow radially from the ganglion. A method of culturing DRG explants where axons grow linearly would greatly assist in the measurement and analysis of growth. Substrates were 3D printed with polylactic Acid (PLA), a biocompatible polymer, using an Ultimaker original printer. Each 60 µm layer of substrate is printed upon the previous one, resulting in grooves or microchannels travelling in parallel across the surface of the print. The substrates were coated with poly D lysine or laminin to improve cell adhesion to the surface. P4 Wistar rat pups were euthanised in accordance with institute guidelines and relevant legislation (directive 2010/63/EU) and their DRGs were cultured on 3D printed substrates in the absence or presence of the antimitotic agent flourodeoxyuridine (FDU) which prevents Schwann cell proliferation. The orientation of the axons was analysed by measuring the angle they projected at relative to the parallel microchannels, and close alignment was seen in both the control and FDU groups (-2.9±1.4°, n=68 and -2.7±1.4°, n=67 respectively; Mean±SEM). This suggests that the axons directly contact and respond to the 3D surface of the microchannel regardless of the presence or otherwise of Schwann cells. By modifying the topography of the substrates, this alignment could also be demonstrated with curved microchannels, further establishing a relationship between the 3D topography of the substrate and the directionality of axon growth. This topographical control of growth can also be combined with molecular cues. DRGs were cultured for 7 days on printed substrates coated with either lysine or laminin. All cultures demonstrated axonal growth and Schwann cell proliferation and migration that closely followed the microchannels, but the addition of laminin extended growth when compared to lysine (5.18±0.60mm; n=6 and 3.52±0.21mm; n=6 respectively; Mean±SD). Topographical cues can independently direct the growth of axons and Schwann cells, where growing neurites somehow sense the spatial gradient provided by microchannels. Varying the surface architecture of 3D prints may offer an efficient and rapid method of studying a regenerating nerves response to a spatial gradient, enabling the design of tissue engineered scaffolds that induce a particular response to a specific topography.

Where applicable, experiments conform with Society ethical requirements