Astrocyte alignment increases neurite outgrowth in a 3D cell culture model

East, Emma; Blum de Oliveira, Daniela; Golding, Jon and Phillips, James (2009). Astrocyte alignment increases neurite outgrowth in a 3D cell culture model. Glia, 57(S13) S159.



Repair of CNS injury is hindered by the glial scar that forms around implanted graft devices. The glial scar is composed of a 3-dimensional (3D) unaligned meshwork of reactive astrocytes that form a physical and physiological barrier, so whilst axons readily enter and traverse the bridging graft, they rarely exit the graft and re-integrate with the host parenchyma. Previous studies have shown that alignment of astrocytes growing in monolayer directs and enhances the growth of neurites over their surface. The aim of this work was to develop a 3D culture system in which the effect of astrocyte alignment on neurite growth could be modelled in a more spatially relevant environment. 3D cell alignment was achieved by co-culturing primary rat astrocytes, neural fibroblasts and DRGs for 3 days in rectangular tethered collagen gels. In this system the cells generate forces that contract the restrained gels, forming a central region in which cells align with the
axis of principal strain. The gels also contain well-defined unaligned areas used as controls. Gels were fixed and stained for GFAP, bIII-tubulin and Hoechst. Astrocyte alignment (aspect ratio) and neurite length were measured and mapped. Astrocyte alignment was significantly greater in the central region of the gels when compared to the control areas (P<0.01). Additionally, neurites in aligned regions grew in an orientated manner following the direction of astrocyte processes, compared to the random orientation of neurite growth in unaligned regions. Furthermore, neurites in aligned regions were significantly longer than those in unaligned areas (P<0.01). Expression of GFAP and neurocan mRNA did not differ between aligned and non-aligned gel regions. In control gels without astrocytes neurites were of similar lengths in both
aligned and unaligned regions. In conclusion, alignment of astrocytes was sufficient to enhance the length and orientation of neurites in a 3D environment, a more clinically relevant model than conventional 2D culture for many investigations of glial-neuronal interaction. This suggests that manipulation of reactive astrocyte alignment in vivo should create a more permissive environment for regenerating axons, and that implantation of bioengineered
devices containing aligned astrocytes may be a promising strategy to promote CNS repair.

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