After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.