(I-336) 3D Printing Astrocytes and ECFCs to Direct Retinal Ganglion Cell Growth and Vascularization of Retinal Constructs

Introduction::

Self-forming retinal organoids have become a valuable tool for the study of retinal development and disease due to their ability to recreate the cell layers of the retina and their ability to respond to light stimuli. However, these organoids cannot be used for the study of glaucoma, the second leading cause of blindness, due to their lacking a properly formed retinal ganglion cell (RGC) nerve fiber layer (NFL). Here we investigate the use of 3D printing to introduce optic nerve head (ONH) astrocytes and endothelial progenitor cells, both of which are not found in retinal organoids but are involved in NFL formation. In addition to RGC guidance, these three cell types are involved in vasculogenesis in vivo, and as such, we investigated the ability of the cells to vascularize the printed retinal constructs.

Materials and Methods:: Primary RGCs and ONH astrocytes were isolated from early postnatal (day 2-5) Sprague Dawley rats with RGCs purified to 99% purity through immunopanning, while endothelial colony forming cells (ECFCs) were isolated from human cord blood mononuclear cells. RGCs and astrocytes were screened through bioinks composed of collagen 1, matrigel and alginate and analyzed for cell survival and spreading. RGC polarization assays were conducted by 3D printing astrocytes onto the center of a radial scaffold, followed by seeding of RGCs surrounding the printed area. Following two days of growth, samples were fixed and stained for βIII-tubulin with RGCs being analyzed for the direction they extend their axon. For astrocyte and ECFC migration experiments, RGCs were seeded on half of the scaffold and astrocytes alone or with ECFCs were positioned at the scaffold center. Samples were cultured for 14 days and stained for βIII-tubulin, S100β and CD31. Samples were analyzed for the percentage of cells migrating on the RGC half of the scaffold and for the distance migrated.

Results, Conclusions, and Discussions::

Results: RGCs and astrocytes were able to survive in collagen 1, matrigel, alginate and mixtures of these three components, however, astrocytes were unable to spread without collagen 1 or matrigel and RGCs were unable to extend neurites outside the presence of matrigel, and as a result, matrigel was used for the 3D printing of astrocytes and ECFCs in further experiments. Astrocytes positioned in the scaffold center increased up to 70% the number of RGCs extending their neurites toward the scaffold center. In both astrocyte and ECFC migration studies, the number of cells migrating on RGC-seeded half of the scaffold was significantly greater than those migrating on the non-seeded side of the scaffold. In addition, astrocytes migrated significantly further on the RGC-seeded side, and all vascularization occurred on the RGC-seeded side of the radial electrospun scaffold.

Discussion and Conclusions: RGC guidance and vascularization in vivo both require the presence of ONH glia and vascular endothelial cells, both of which are absent in the self-forming retinal organoids. In this study, we demonstrate that introducing these cells via 3D printing in combination with electrospun scaffolds is able to polarize RGC growth such that it more closely matches that found in vivo while also demonstrating that these three cell types work in combination to recreate the mechanism of vascular formation during retinal development. These results suggest a method for forming retinal organoids that can better be used for studying glaucoma while also demonstrating a potential method for vascularizing CNS tissue engineered constructs.

Acknowledgements (Optional): : We would like to acknowledge the support from the National Eye Institute (R01-EY028946 KEK) and support from RPB 2018 Research to Prevent Blindness/Stavros Niarchos Foundation International Research Collaborators Award.

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