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Ivan Fernandez-Bueno, David Rodriguez-Crespo, Salvatore Di Lauro, Amar K Singh, Maite Garcia-Gutierrez, Manuel Garrosa-García, Jose-Carlos Pastor; A Triple-layer Co-culture Model Of Neuroretina, RPE And Adipose-MSCs To Evaluate Cell Therapy In Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4001.
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To develop and standardize a co-culture model with three layers, neuroretina, retinal pigment epithelium (RPE) and adipose-derived mesenchymal stem cells (AD-MSCs), for evaluating AD-MSCs potential in retinal degeneration
This study followed the tenets of the Declaration of Helsinki and was approved by the IRC of the University of Valladolid (Spain). RPE cells from porcine eyes and AD-MSCs from human lipo-aspirates were isolated, maintained in culture and characterized as previously described by our group (Srivastava et al., 2011; Singh et al., 2013). RPE cells were seeded (30,000 cells/cm2) on the bottom of Transwell culture plates. AD-MSCs were seeded (30,000 cells/cm2) on the inferior surface of Transwell membranes (TWM). TWM with growing AD-MSCs on its inferior surface were inserted into culture plates with growing RPE cells on its bottom surface. Then, 5x5 mm neuroretinal explants were obtained from porcine eyes, as previously described by our group (Fernandez-Bueno et al. 2008), and explanted on the superior surface of the TWM. These three physically separated cellular layers were co-cultured in Neurobasal A/DMEM (1:1) medium during 6 days. In parallel, cells and neuroretinas were individually culture as controls. RPE and AD-MSCs viability, proliferation and/or phenotype were determined. Cryostat neuroretina sections were immunostained to evaluate retinal degeneration process
RPE and AD-MSCs adequately survived and proliferated in the experimental condition. AD-MSCs showed significantly decreased alpha-SMA (fibroblasts) and increased recoverin (photoreceptors) immunoexpression, while CK8/18 (epithelial) and nestin (stem cells) immunoexpressions were similar to respective controls. Neuroretina immunoexpression of GFAP and CRALBP (reactive gliosis) in the experimental condition was similar to spontaneously degenerated controls at 6 days
The triple-layer co-culture model developed, not previously described, could mimic an ex vivo subretinal space to evaluate cell therapy potential in retinal degeneration. In this model, AD-MSCs potentially expressed retinal cell proteins, however, were unable to preserve retinal tissue from spontaneous degeneration. This finding suggests that external biomolecules are necessary to reinforce AD-MSCs neuroprotective effects on degenerated neuroretina
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