Three-Dimensional Bioprinting of Astrocytes and Endothelial Cells to Direct Retinal Axon Growth and Vascularization.

Retinal organoids (ROs) are currently used to study retinal development and diseases but cannot model glaucoma because they fail to form a nerve fiber layer (NFL) and optic nerve (ON). Utilizing three-dimensional bioprinting, ON head astrocytes (ONHAs) and vascular endothelial cells, both of which contribute to NFL development in vivo but are absent in ROs, were positioned at the center of scaffolds seeded with retinal ganglion cells (RGCs). In experiments using ONHAs isolated from developing retinas, polarization of RGC neurite growth increased by 43% while ONHA from adult retinas or astrocytes from the developing peripheral retina or developing cortex did not increase polarization above controls. Furthermore, RGC-seeded scaffolds increased both the number and rate of ONHAs migrating out from the printed center compared to scaffolds lacking RGCs, mimicking the migration pattern observed during retinal development. Finally, in scaffolds containing both ONHAs and endothelial cells, the endothelial cells preferentially migrate on and only form vascular tube structures on scaffolds also containing RGCs. These results suggest that recreating the developmental organization of the retina can recapitulate the mechanism of NFL development and retinal vascularization in vitro. This step is not only necessary for the development of retinal models of glaucoma but has the potential for translation to other parts of the central nervous system.