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T. V. Johnson, K. R. Martin; Development and Characterization of Adult Retinal Explant Organotypic Tissue Culture as an in vitro Model for Intravitreal Stem Cell Transplantation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4286.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of stem/progenitor cells to treat retinal neurodegenerative disease is a subject of intense investigation. Current therapeutic efforts are limited by low numbers of cells integrating into the retina and suboptimal control over the differentiation and behaviour of grafts. To facilitate investigation into novel methods of improving retinal stem cell therapy, we developed a retinal explant culture system using adult rats.
Retinas were explanted from 8-12 week old Sprague Dawley rats retinal ganglion cell (RGC) side up on Millipore filters in B27/N2-supplemented serum-free media or in media containing 25% normal horse serum (NHS) for up to 17 days. Tissue viability was assessed at various time points by gross morphology, propidium iodide (PI) uptake, quantification of cell survival, activated caspase-3 expression, and protein expression patterns. To model intravitreal cell transplantation, 1-2x103 human Müller progenitor cells (hMIO-M1) in 2µL of media were placed on explants. Explant morphology and immunohistochemistry were compared to sectioned whole eyes with or without prior intravitreal hMIO-M1 transplantation.
Explants cultured in B27/N2 media were viable through 17 days as evidenced by PI exclusion, static cell densities, consistently low caspase-3 expression and little morphological change. In contrast, NHS media was associated with obvious tissue degradation beginning peripherally and encompassing 73±4% (mean ± SEM) of the tissue by day 17; greater and more diffuse PI uptake; significant cell loss over time, especially from the RGC layer (77±5 on day 3 vs. 14±2 cells/mm on day 14, p<0.01); and a temporal increase in active caspase-3+ cells (4±1% on day 3 vs. 19±5% on day 17 in the RGC layer, p<0.01). Explants in B27/N2 media were strongly immunoreactive for β-III-tubulin, neurofilament, NeuN, Brn3a, and Thy-1 as well as GFAP, vimentin, nestin, and glutamine synthetase in the inner retina, whereas expression was weak for NHS media and decreased with time. Seven days after transplantation, glial reactivity as assessed by GFAP expression was highly upregulated in explants and control eyes. Some grafted cells migrated into the retina, but the majority remained outside of the inner limiting membrane.
Retinal explants cultured in B27/N2 media are viable for at least 2 weeks and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to integrate. This system will be useful for investigating methods to enhance retinal stem cell therapy by providing a manipulatable in vitro model.
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