Kador, Karl1; Abukunna, Fatima1
1Department of Ophthalmology, University of Missouri-Kansas City
Purpose: Self-forming retinal organoids (RO) have become a valuable tool in the study of retinal degenerative diseases as well as source for cell replacement therapies due to their ability to recreate all the cells of the neural retina in their correct cell lamina. However, these RO fail to create a properly organized retinal nerve fiber layer (NFL) or optic nerve (ON) due to the retinal ganglion cells (RGC) growing out from the RO, limiting their ability to be used as a model of glaucoma. In addition, these RO lack vasculature limiting their use as a model for diseases such as diabetic retinopathy. Interestingly, during development, the formation of the NFL, the ON and vasculature in the neural retina are linked in a common mechanism in which ON cells secrete factors which polarize the RGC growth before the ON head astrocytes (ONHA) migrate along the RGC axons while secreting VEGF to direct vascular cell migration. In this study, we use 3D bioprinting to recreate this mechanism in vitro.
Methods: RGCs were isolated from early postnatal Sprague Dawley rats (postnatal day 2-5) and purified through a two step immunopanning protocol.
ONHA were isolated from postnatal day 2 (P2) and adult rats by dissecting the ON from an intact retina. Retinal astrocytes were isolated from P2 rats. Cells were used between passage 3-6.
RGC Polarization Assays: Astrocytes were suspended in matrigel and printed using an Allevi 3 bioprinter at the center of radial electrospun scaffolds. RGCs were seeded the following day and cultured for 2 days before fixing. Samples were analyzed for the direction the RGCs extend their axon.
Astrocyte and ECFC Migration Assays: RGCs were seeded on half of the radial scaffold and P2 ONHA alone or with endothelial colony forming cells (ECFC) were positioned at the scaffold center. Samples were cultured for 14 days, fixed and stained. Samples were analyzed for the percentage of cells migrating on the RGC half of the scaffold compared to those on the fibers alone half.
Results: RGCs seeded on scaffolds with developmental P2 ONHA were observed to have an increased polarization of axon growth towards the scaffold center where as RGCs on scaffolds with adult ONHA, P2 retinal astrocytes or two cortical astrocyte cell lines derived from P2 mice showed no change in polarization compared to matrigel printed alone. A higher percentage of ONHA were observed to migrate on the half of the scaffold seeded with RGCs with those ONHA migrating a further distance as well. Similarly, ECFCs were observed to preferentially migrate on the RGC half of the scaffold, however while some ECFCs were observed to migrate on the non-RGC half of the scaffold, vascular tubes were only observed to form on the half of the scaffold which contained RGCs.
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 RO. In this study, we demonstrate that introducing astrocytes from a specific developmental time point via 3D printing in combination with electrospun scaffolds are able to polarize RGC growth such that it more closely matches that found in vivo while also demonstrating that these three cell types recapitulate the mechanism for retinal vascularization. These results suggest a method for forming RO that can better be used for studying glaucoma while also demonstrating a potential method for vascularizing CNS tissue engineered constructs.
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