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Karen Peynshaert, Stefaan De Smedt, Kevin Braeckmans, Katrien Remaut; Study into the role of Müller cells in nanoparticle-based retinal gene therapy after intravitreal injection. Invest. Ophthalmol. Vis. Sci. 2016;57(12):784. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The vitreoretinal interface is one of the primary barriers that hinders the delivery and thus efficiency of non-viral gene vectors that have the retina as their target. Interestingly, several groups have proposed that Müller cells might act as our gateway to shuttle intravitreally injected particles across this barrier. We therefore aim to develop an ex vivo retinal model that, in contrast to existing models, is bovine-derived and keeps the vitreous and vitreoretinal interface intact. Various nanomedicines can then be injected ex vivo in the vitreous, after which their route inside the retina can be identified.
Bovine eyes are obtained from the abattoir. The retina – with vitreous attached - is detached from the RPE and placed on a Transwell® filter for culture. MitoTracker Deep Red (10 µM), FM 1-43 (20 µg/ml) and carboxylated polystyrene particles are intravitreally injected and incubated overnight to allow diffusion from above. A z-stack of confocal images (Nikon C1si confocal microscope) of the retina is then recorded using a Nikon 60x NIR Apo water dipping objective that is brought into contact with the vitreous layer on top of the retina.
The Mitotracker staining of the explant confirms that the ganglion cell layer is virtually completely occupied by Müller cell endfeet. Moreover, when scanning the literature we found that, unlike porcine and rodent species, this layer in bovine eyes greatly resembles human physiology. The lipid dye FM 1-43 outlines every Müller cell in this layer and clearly stains veins running through the retina. Following intravitreal injection of polystyrene particles, some colocalize with Müller cells, suggesting these cells could form a transport route across the retina. However, the distance of injection from the retina and nanoparticle concentration should be optimized.
We developed a model that maximally mimics human ocular physiology. We suggest that this model can serve as a set-up to investigate the influence of particle physicochemistry on their ability to cross the vitreoretinal interface after intravitreal injection. In the near future, we will evaluate the uptake and cellular trafficking of these particles in vitro in human spontaneously immortalized Müller cells to investigate intracellular barriers.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Ganglion cell layer with Müller cell endfeet (red), lipid membranes and veins (green). Scale bar = 20µm.
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