Abstract
Purpose :
The use of retinal organoids (RO) for the study of glaucoma is limited due to their lack of a properly formed nerve fiber layer (NFL). In vivo, optic nerve head astrocytes (ONHA) and vascular cells secrete factors to direct retinal ganglion cell (RGC) growth but are absent in RO. Here we use 3D printing combined with biodegradable scaffolds to introduce ONHA and endothelial colony forming cells (ECFCs) to direct RGC growth. In addition to RGC guidance, these three cell types are also involved in vasculogenesis, and as such we investigated the ability of the of the cells to vascularize the printed retinal constructs.
Methods :
RGCs and ONHA were isolated from postnatal rats (day 2-5) and ECFCs were isolated from human cord blood mononuclear cells. Bioinks composed of collagen 1, matrigel and alignate were screened with both RGCs and ONHA and analyzed for cell survival and spreading. RGC polarization assays were conducted by 3D printing ONHA onto the center of a radial scaffold followed by seeding of RGCs surrounding the printed area and cultured for two days. Samples were stained and analyzed for the direction RGCs extend their axons. To measure ONHA and ECFC migration, RGCs were seeded on half of the scaffold and ONHA alone or with ECFCs were positioned at the scaffold center and 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 :
RGCs and ONHA were able to survive in in all bioinks, however ONHA were unable to spread without collagen 1 or matrigel and RGCs were unable to extend neurites without matrigel. 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 scaffold.
Conclusions :
Using 3D printing to introduce ONHA and ECFCs into retinal constructs can polarize RGC growth in vitro, while also reproducing the mechanism of vasculogenesis observed during retinal development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.