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Arantxa Bolinches-Amorós, Tina storm, Angela J. Russell, Stephen G Davies, Alun R Barnard, Robert E MacLaren; Optimization of an in vitro model for assessing the survival of post-mitotic photoreceptors and Müller glial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4600.
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© ARVO (1962-2015); The Authors (2016-present)
Although primary retinal cell cultures lack the 3D organization present in vivo or in retinal explants, these primary cultures are a very valuable in vitro model for the study of the biology of concrete retinal cell types.While dissociated retinal cells from perinatal mice survive well in culture, those from post-natal ages above day 10 have a decreased survival in vitro. The aim of this study was develop an in vitro model of primary post-mitotic retinal cell cultures with an increased survival.
Harvesting and enzymatic (papain) dissociation of retinas from Nrl-GFP reporter mice was performed at different post-natal ages. The duration of papain incubation was also systematically varied.Given the high proportion of rod photoreceptors in the retina, the removal of this rod fraction allows for the clearer observation and study of other retinal cell types, such as Müller glial cells. Thus, separation of the rod fraction from the mixed population of dissociated retinal cells was performed by Magnetic Activated Cell Sorting (MACS) against CD73. CD73-positive (rod enriched), CD73-negative (rod depleted) and native, unsorted dissociated retinal cells were cultured separately. The influence of cultureware surface coating was also explored.Rod photoreceptors were monitored by the presence of the reporter gene, EGFP. Müller glial cells were identified in the primary cultures by morphology and immunostaining.
Various factors affected the survival of photoreceptors and Müller glia in culture after tissue dissociation and subsequent MACS. Adjusting the incubation time with papain around 15 minutes ensures the release of Müller glia and a more gentle dissociation for the photoreceptors, thus preserving their integrity. Seeding the cells on a poly-D-lysine treated vs standard adherent surface increases the attachment and subsequent survival of Müller glia after the retinal dissociation and MACS.
We have optimized a protocol for increasing the survival of cells after retina dissociation followed by MACS to separate the fraction of rod photoreceptors from the rest of retinal cells.This protocol provides an efficient in vitro model to assess the survival of post-mitotic rod photoreceptors and Müller glial cells, as well as a valuable model for screening compounds affecting the survival of these retinal cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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