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Jonathan Pieter Vande Geest, Jr-Jiun Liou, Ashlinn M Sweeney, Michelle Drewry, Bryan N Brown; A decelluarization and recellularization platform to study optic nerve head remodeling. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2221.
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Identifying the precise time course and dynamics of lamina cribrosa (LC) remodeling in glaucoma is challenging. In vitro cell-seeded scaffolds often fail to recapitulate the optic nerve head (ONH) microenvironment due to the oversimplified geometry and nonphysiological matrix stiffness. While most common rodent models may be suitable for studying molecular mechanisms, these animals lack the collagenous LC present in humans. Large animals do contain a collagenous LC however the cost of these experiments limits high throughput experimentation. Given these limitations, we aim to develop an ex-vivo platform that utilizes human cells and human ONHs for studying LC mechanobiology that are high throughput and allow controlled biomechanical and genetic manipulation. In this study, we want to demonstrate that we can decellularize and recellularize ONHs in a controlled manner.
Porcine ONHs were decellularized using a detergent and enzyme-based protocol as previously described. Decellularized ONHs were seeded with porcine LC cells in polyethylene glycol (PEG), methacrylated gelatin (GelMA), or methacrylated collagen (ColMA) hydrogels. Recellularized ONHs were cultured and stained with live/dead assay at days 1, 3, and 7 (n=3 per group per time point). One-way ANOVA with Tukey’s post hoc was performed for group comparison.
DNA quantification and histology confirmed that decellularization reduced the cellular content of the tissue by 94.9% compared to native tissue (p=0.002) while preserving the ECM microstructure and basement membrane of the matrix. PEG 6000 and PEG 10000 hydrogel infiltration were able to restore the biomechanical response of the decellularized ONH, having normalized LC displacements of 0.68±0.5 (p=0.36) and 0.87±0.4 (p=0.96), respectively. During recellularization, percentage of live cells (green) is higher in GelMA and ColMA hydrogels when compared to PEG hydrogels (Figure 1).
We successfully established a decellularization and recellularization platform using porcine optic nerve heads. We are currently investigating whether ONH cells directly influence retinal ganglion cell health in a co-culture system with rat retinal ganglion cells. This platform will enable us to use human cells and tissues in future research where we will understand how tissue stiffness and genetics drive LC remodeling in human tissues and potentially for glaucoma drug testing.
This is a 2020 ARVO Annual Meeting abstract.
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