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Salvatore Di Lauro, Ivan Fernandez-Bueno, David Rodríguez-Crespo, Girish K Srivastava, Amar K Singh, Maite Garcia-Gutierrez, Manuel Gayoso, Jose-Carlos Pastor; An organotypic culture model of porcine neuroretina supplemented with porcine retinal pigment epithelium (pRPE) cells to simulate an ex vivo subretinal space. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6314.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a new physically separated co-culture model of neuroretina with retinal pigment epithelium (RPE) cells that simulates an ex vivo subretinal space to recreate some of the conditions that follow retinal detachment.
RPE cells from porcine eyes (pRPE) were isolated and maintained in culture as previously described by our group (Srivastava et al., 2011). pRPE cells were seeded (30,000 cells/cm2) on the bottom of Transwell culture plates for 24 hrs for adherence and growth. Neuroretina explants (5x5 mm), from the porcine area centralis, were obtained as previously described by our group (Fernandez-Bueno et al. 2008). Explants were cultured over Transwell membranes with the photoreceptor layer facing the supporting membrane. Neuroretina explants supplemented with pRPE cells were co-cultured in Neurobasal A/DMEM (1:1) medium during 9 days. In parallel, neuroretina explants were cultured alone as controls. Specimens were processed for epoxy-resin embedding and cryosectioning. Light and immunofluorescence microscopy were performed, using toluidine blue staining and antibodies against GFAP, CRALBP, calbindin (CB) and synaptophysin (SYP) to evaluate retinal modifications. Specimen thickness was evaluated over neuroretinal images using the software Image J (version 1.47v, NIH Image, National Institute of Health, EE.UU).
Neuroretina explants co-cultured with pRPE better preserved tissular architecture and cellular organization than controls at 9 days of culture. Co-cultured explants thickness was statistically significant maintained than in controls after culture (109,35±4,24 μm vs 92,28±14,94 μm; p<0.05). Protein immunoexpressions in co-cultures revealed less reactive gliosis (GFAP and CRALBP) and preserved cones (CB) and synapses (SYP) compared to controls.
A double-layer co-culture model of neuroretina and RPE cells was successfully developed and standardized. In this model, RPE cells presence reduced retinal degeneration changes, probably related to the secretion of neuroprotective factors by RPE cells. Physically separation between neuroretina and RPE could recreate an ex vivo subretinal space. Thus, this model may be useful to evaluate potential therapies for retinal detachment and other retinal degeneration processes.
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